BICYCLIC PEPTIDE LIGANDS SPECIFIC FOR EphA2

ABSTRACT

The present invention relates to polypeptides which are covalently bound to non-aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of the Eph receptor tyrosine kinase A2 (EphA2). The invention also includes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups, to pharmaceutical compositions comprising said peptide ligands and drug conjugates and to the use of said peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No/ 16/220,685, filed Dec. 14,2018, which claims the benefit of GB Application No. 1721259.8, filedDec. 19, 2017, GB Application No.

1804102.0, filed Mar. 14, 2018, and GB Application No. 1818603.1, filedNov. 14, 2018, the entirety of each of which is hereby incorporated byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Sep. 22, 2022, isnamed Bicycle_193449_SL.xml and is 14,754 bytes in size.

FIELD OF THE INVENTION

The present invention relates to polypeptides which are covalently boundto non-aromatic molecular scaffolds such that two or more peptide loopsare subtended between attachment points to the scaffold. In particular,the invention describes peptides which are high affinity binders of theEph receptor tyrosine kinase A2 (EphA2). The invention also includesdrug conjugates comprising said peptides, conjugated to one or moreeffector and/or functional groups, to pharmaceutical compositionscomprising said peptide ligands and drug conjugates and to the use ofsaid peptide ligands and drug conjugates in preventing, suppressing ortreating a disease or disorder characterised by overexpression of EphA2in diseased tissue (such as a tumour).

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and targetspecificity to protein targets and hence are an attractive moleculeclass for the development of therapeutics. In fact, several cyclicpeptides are already successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008),Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result froma relatively large interaction surface formed between the peptide andthe target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), acyclic peptide with the Arg-Gly-Asp motif binding to integrin aVb3 (355Å²) (Xiong et al. (2002),

Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1binding to urokinase-type plasminogen activator (603 Å²; Zhao et al.(2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8, (MMP-8) which lost its selectivity over other MMPswhen its ring was opened (Cherney et al. (1998), J Med Chem 41 (11),1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin andactinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp and McNamara(1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen andco-workers had used tris(bromomethyl)benzene and related molecules forrapid and quantitative cyclisation of multiple peptide loops ontosynthetic scaffolds for structural mimicry of protein surfaces(Timmerman et al. (2005), ChemBioChem). Methods for the generation ofcandidate drug compounds wherein said compounds are generated by linkingcysteine containing polypeptides to a molecular scaffold as for exampleTATA (1,1′,1″-(1,3,5-triazinane-1,3,5-triyhtriprop-2-en-1-one, Heinis etal. Angew Chem, Int Ed. 2014; 53:1602-1606).

Phage display-based combinatorial approaches have been developed togenerate and screen large libraries of bicyclic peptides to targets ofinterest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009/098450). Briefly, combinatorial libraries of linear peptidescontaining three cysteine residues and two regions of six random aminoacids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclisedby covalently linking the cysteine side chains to a small moleculescaffold.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided apeptide ligand specific for EphA2 comprising a polypeptide comprising atleast three cysteine residues, separated by at least two loop sequences,and a non-aromatic molecular scaffold which forms covalent bonds withthe cysteine residues of the polypeptide such that at least twopolypeptide loops are formed on the molecular scaffold, wherein thepeptide ligand comprises the amino acid sequence:

-   -   C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii) (SEQ ID NO: 1);

wherein HyP is hydroxyproline, HArg is homoarginine and C_(i), C_(ii)and C_(iii) represent first, second and third cysteine residues,respectively or a pharmaceutically acceptable salt thereof.

According to a further aspect of the invention, there is provided a drugconjugate comprising a peptide ligand as defined herein conjugated toone or more effector and/or functional groups.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand or a drugconjugate as defined herein in combination with one or morepharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided apeptide ligand or drug conjugate as defined herein for use inpreventing, suppressing or treating a disease or disorder characterisedby overexpression of EphA2 in diseased tissue (such as a tumour).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : General schematic demonstrating the concept of preparingBicycle drug conjugates (BDCs).

FIG. 2 : Plot of mean tumour volume versus time for BCY6136 in HT1080xenograft mice. Doses (2, 3 and 5 mg/kg) were administered on days 0 and7. Body weight changes during treatment indicative of tumour burden,drug-associated toxicology and overall animal health are illustrated inthe top right inset.

FIG. 3 : Plot of mean tumour volume versus time for BCY6136 in NCI-H1975xenograft mice. Doses (1, 2 and 3 mg/kg) were administered on days 0, 7,14, 21, 28 and 35. Body weight changes during treatment indicative oftumour burden, drug-associated toxicology and overall animal health areillustrated in the top right inset.

FIG. 4 : Plot of mean tumour volume versus time for BCY6136 inMDA-MB-231 xenograft mice. Doses (1, 2 and 3 mg/kg) were administered onday 0, 7, 14, 21, 28, 35 and 45. Body weight changes during treatmentindicative of tumour burden, drug-associated toxicology and overallanimal health are illustrated in the top right inset.

FIGS. 5 and 6 : Body weight changes and tumor volume traces afteradministering BCY6136 (FIG. 5 ) and ADC (FIG. 6 ) to female BALB/c nudemice bearing PC-3 xenograft. Data points represent group mean bodyweight.

FIG. 7 : Body weight changes and tumor volume traces after administeringBCY6136, EphA2-ADC or Docetaxel to male Balb/c nude mice bearing PC-3xenograft. Data points represent group mean body weight.

FIG. 8 : Body weight changes and tumor volume trace after administeringBCY6136 to female Balb/c nude mice bearing NCI-H1975 xenograft. Datapoints represent group mean tumor volume and body weight.

FIGS. 9 and 10 : Body weight changes and tumor volume traces afteradministering BCY6136 and ADC to female Balb/c nude mice bearingLU-01-0251 xenograft. Data points represent group mean body weight.

FIG. 11 : Body weight changes and tumor volume traces afteradministering BCY6136 to female Balb/c nude mice bearing LU-01-0046.Data points represent group mean body weight.

FIG. 12 : Body weight changes and tumor volume traces afteradministering BCY6136 or ADC to female Balb/c nude mice bearingLU-01-0046 NSCLC PDX model. Data points represent group mean bodyweight.

FIGS. 13 to 15 : Body weight changes and tumor volume traces afteradministering BCY6136 (FIG. 13 ), BCY6173 (FIG. 14 ) and BCY6175 (FIG.15 ) to female Balb/c nude mice bearing LU-01-0046. Data pointsrepresent group mean body weight.

FIG. 16 : Body weight changes and tumor volume traces afteradministering BCY6136 (referred to in FIG. 16 as BT5528), BCY8245 orBCY8781 to female BALB/c nude mice bearing LU-01-0412 xenograft. Datapoints represent group mean tumor volume (left panel) and body weight(right panel).

FIG. 17 : Body weight changes and tumor volume traces afteradministering BCY6136 to female Balb/c nude mice bearing LU-01-0486xenograft. Data points represent group mean body weight.

FIG. 18 : Body weight changes and tumor volume trace after administeringBCY6136 to female Balb/c nude mice bearing MDA-MB-231-luc xenograft.Data points represent group mean tumor volume and body weight.

FIG. 19 : Body weight changes and tumor volume traces afteradministering BCY6136 to female BALB/c mice bearing EMT-6 syngeneic.Data points represent group mean body weight. The dosage of group 3 andgroup 4 was changed to 5 mpk and 3 mpk from Day 14.

FIG. 20 : Body weight changes and tumor volume traces afteradministering BCY6136 to female Balb/c nude mice bearing NCI-N87xenograft. Data points represent group mean body weight.

FIG. 21 : Body weight changes and tumor volume traces afteradministering BCY6136 to female Balb/c nude mice bearing SK-OV-3xenograft. Data points represent group mean body weight.

FIG. 22 : Body weight changes and tumor volume traces afteradministering BCY6136 to female Balb/c nude mice bearing OE21 xenograft.Data points represent group mean body weight.

FIG. 23 : Body weight changes and tumor volume traces afteradministering BCY6136 to female CB17-SCID mice bearing MOLP-8 xenograft.Data points represent group mean body weight.

FIGS. 24 to 29 : Body weight changes and tumor volume traces afteradministering BCY6173 (FIG. 24 ), BCY6135 (FIG. 25 ), BCY6136 (FIG. 26), BCY6174 (FIG. 27 ), BCY6175 (FIG. 28 ) and ADC (FIG. 29 ) to femaleBALB/c nude mice bearing HT1080 xenograft. Data points represent groupmean body weight.

Where error bars are present in the above Figures, these representstandard error of the mean (SEM).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the peptide ligand comprises the amino acid sequence:

(SEQ ID NO: 2) (β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii )(BCY6099);

wherein Sar is sarcosine, HArg is homoarginine and HyP ishydroxyproline.

In one embodiment, the molecular scaffold is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

In a further embodiment, the molecular scaffold is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and thepeptide ligand comprises the amino acid sequence:

(SEQ ID NO: 2) (β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii) (BCY6099);and

wherein Sar is sarcosine, HArg is homoarginine and HyP ishydroxyproline.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4thed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

NOMENCLATURE

Numbering

When referring to amino acid residue positions within the peptides ofthe invention, cysteine residues (C_(i), C_(ii) and C_(iii)) are omittedfrom the numbering as they are invariant, therefore, the numbering ofamino acid residues within the peptides of the invention is referred toas below:

(SEQ ID NO: 1) -C_(i)-HyP₁-L₂-V₃-N₄-P₅-L₆-C_(ii)-L₇-H₈-P₉-(D-Asp)₁₀-W₁₁-(HArg)₁₂-C_(iii)-.

For the purpose of this description, all bicyclic peptides are assumedto be cyclised with1,1′,1″-(1,3,5-triazinane-1,3,5-triyhtriprop-2-en-1-one (TATA) yieldinga tri-substituted 1,1′,1″-(1,3,5-triazinane-1,3,5-triyhtripropan-1-onestructure. Cyclisation with TATA occurs on C_(i), C_(ii) and C_(iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added tothe left or right side of the sequence, separated by a hyphen. Forexample, an N-terminal ((3-Ala)-Sarco-Ala tail would be denoted as:

(SEQ ID NO: X) (β-Ala)-Sar₁₀-A-.

Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3),1362-1373, it is envisaged that the peptide sequences disclosed hereinwould also find utility in their retro-inverso form. For example, thesequence is reversed (i.e. N-terminus becomes C-terminus and vice versa)and their stereochemistry is likewise also reversed (i.e. D-amino acidsbecome L-amino acids and vice versa).

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide, peptidicor peptidomimetic covalently bound to a molecular scaffold. Typically,such peptides, peptidics or peptidomimetics comprise a peptide havingnatural or non-natural amino acids, two or more reactive groups (i.e.cysteine residues) which are capable of forming covalent bonds to thescaffold, and a sequence subtended between said reactive groups which isreferred to as the loop sequence, since it forms a loop when thepeptide, peptidic or peptidomimetic is bound to the scaffold. In thepresent case, the peptides, peptidics or peptidomimetics comprise atleast three cysteine residues (referred to herein as C_(i), C_(ii) andC_(iii)), and form at least two loops on the scaffold.

Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number ofadvantageous properties which enable them to be considered as suitabledrug-like molecules for injection, inhalation, nasal, ocular, oral ortopical administration. Such advantageous properties include:

Species cross-reactivity. This is a typical requirement for preclinicalpharmacodynamics and pharmacokinetic evaluation;

Protease stability. Bicyclic peptide ligands should in mostcircumstances demonstrate stability to plasma proteases, epithelial(“membrane-anchored”) proteases, gastric and intestinal proteases, lungsurface proteases, intracellular proteases and the like. Proteasestability should be maintained between different species such that abicyclic peptide lead candidate can be developed in animal models aswell as administered with confidence to humans;

Desirable solubility profile. This is a function of the proportion ofcharged and hydrophilic versus hydrophobic residues andintra/inter-molecular H-bonding, which is important for formulation andabsorption purposes;

An optimal plasma half-life in the circulation. Depending upon theclinical indication and treatment regimen, it may be required to developa bicyclic peptide with short or prolonged in vivo exposure times forthe management of either chronic or acute disease states. The optimalexposure time will be governed by the requirement for sustained exposure(for maximal therapeutic efficiency) versus the requirement for shortexposure times to minimise toxicological effects arising from sustainedexposure to the agent.

Selectivity. Certain peptide ligands of the invention demonstrate goodselectivity over other Eph receptor tyrosine kinases, such as EphA1,EphA3, EphA4, EphA5, EphA6, EphA7, EphB1, factor XIIA, carbonicanhydrase 9 and CD38 (selectivity data for selected peptide ligands ofthe invention may be seen in Tables 11 and 12). It should also be notedthat selected peptide ligands of the invention exhibit cross reactivitywith other species (e.g. mouse and rat) to permit testing in animalmodels (Tables 3, 7-8, 10 and 12); and

Safety. Bleeding events have been reported in pre-clinical in vivomodels and clinical trials with EphA2 Antibody Drug Conjugates. Forexample, a phase 1, open-label study with

MEDI-547 was halted due to bleeding and coagulation events that occurredin 5 of 6 patients (Annunziata et al, Invest New Drugs (2013) 31:77-84).The bleeding events observed in patients were consistent with effects onthe coagulation system observed in rat and monkey pre-clinical studies:increased activated partial thromboplastin time and increasedfibrinogen/fibrin degradation product (Annunziata et al IBID). Overtbleeding events were reportedly seen in toxicology studies in monkeys(Annunziata et al, IBID). Taken together these results imply thatMEDI-547 causes Disseminated Intravascular Coagulation (DIC) in bothpreclinical species and patients. The BDCs reported here have short invivo half lives (<30 minutes) and are therefore intrinsically lesslikely to give rise to DIC in patients. Results shown here (seeBIOLOGICAL DATA sections 5 and 6 and Table 15) demonstrate that selectedBicycle Drug Conjugates of the invention have no effect on coagulationparameters and gave rise to no bleeding events in pre-clinical studies.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of thisinvention, and references to peptide ligands include the salt forms ofsaid ligands.

The salts of the present invention can be synthesized from the parentcompound that contains a basic or acidic moiety by conventional chemicalmethods such as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. One particular salt is the hydrochloride salt.Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO⁻), then a salt may be formed with anorganic or inorganic base, generating a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R^(+, NH) ₂R₂ ⁺, NHR₃ ⁺, NR₄⁺). Examples of some suitable substituted ammonium ions are thosederived from: methylamine, ethylamine, diethylamine, propylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the peptides of the invention contain an amine function, these mayform quaternary ammonium salts, for example by reaction with analkylating agent according to methods well known to the skilled person.Such quaternary ammonium compounds are within the scope of the peptidesof the invention.

Isotopic Variations

The present invention includes all pharmaceutically acceptable(radio)isotope-labeled peptide ligands of the invention, wherein one ormore atoms are replaced by atoms having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberusually found in nature, and peptide ligands of the invention, whereinmetal chelating groups are attached (termed “effector”) that are capableof holding relevant (radio)isotopes, and peptide ligands of theinvention, wherein certain functional groups are covalently replacedwith relevant (radio)isotopes or isotopically labelled functionalgroups.

Examples of isotopes suitable for inclusion in the peptide ligands ofthe invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T),carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, suchas ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³N and¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur,such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga,yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies, and to clinically assessthe presence and/or absence of the EphA2 target on diseased tissues. Thepeptide ligands of the invention can further have valuable diagnosticproperties in that they can be used for detecting or identifying theformation of a complex between a labelled compound and other molecules,peptides, proteins, enzymes or receptors. The detecting or identifyingmethods can use compounds that are labelled with labelling agents suchas radioisotopes, enzymes, fluorescent substances, luminous substances(for example, luminol, luminol derivatives, luciferin, aequorin andluciferase), etc.

The radioactive isotopes tritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C,are particularly useful for this purpose in view of their ease ofincorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention cangenerally be prepared by conventional techniques known to those skilledin the art or by processes analogous to those described in theaccompanying Examples using an appropriate isotopically-labeled reagentin place of the non-labeled reagent previously employed.

Non-Aromatic Molecular Scaffold

References herein to the term “non-aromatic molecular scaffold” refer toany molecular scaffold as defined herein which does not contain anaromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system.

Suitable examples of non-aromatic molecular scaffolds are described inHeinis et al (2014)

Angewandte Chemie, International Edition 53(6) 1602-1606.

As noted in the foregoing documents, the molecular scaffold may be asmall molecule, such as a small organic molecule.

In one embodiment the molecular scaffold may be a macromolecule. In oneembodiment the molecular scaffold is a macromolecule composed of aminoacids, nucleotides or carbohydrates.

In one embodiment the molecular scaffold comprises reactive groups thatare capable of reacting with functional group(s) of the polypeptide toform covalent bonds.

The molecular scaffold may comprise chemical groups which form thelinkage with a peptide, such as amines, thiols, alcohols, ketones,aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides,anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

An example of an a unsaturated carbonyl containing compound is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)(Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

Effector and Functional Groups

According to a further aspect of the invention, there is provided a drugconjugate comprising a peptide ligand as defined herein conjugated toone or more effector and/or functional groups.

Effector and/or functional groups can be attached, for example, to the Nand/or C termini of the polypeptide, to an amino acid within thepolypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragmentsthereof. For instance, an effector group can include an antibody lightchain constant region (CL), an antibody CH1 heavy chain domain, anantibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, orany combination thereof, in addition to the one or more constant regiondomains. An effector group may also comprise a hinge region of anantibody (such a region normally being found between the CH1 and CH2domains of an IgG molecule).

In a further embodiment of this aspect of the invention, an effectorgroup according to the present invention is an Fc region of an IgGmolecule. Advantageously, a peptide ligand-effector group according tothe present invention comprises or consists of a peptide ligand Fcfusion having a tβ half-life of a day or more, two days or more, 3 daysor more, 4 days or more, 5 days or more, 6 days or more or 7 days ormore. Most advantageously, the peptide ligand according to the presentinvention comprises or consists of a peptide ligand Fc fusion having atβ half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactivegroups for the attachment of other entities, functional groups which aiduptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptidesagainst intracellular targets to be effective. Targets that can beaccessed by peptides with the ability to penetrate into cells includetranscription factors, intracellular signalling molecules such astyrosine kinases and molecules involved in the apoptotic pathway.Functional groups which enable the penetration of cells include peptidesor chemical groups which have been added either to the peptide or themolecular scaffold. Peptides such as those derived from such as VP22,HIV-Tat, a homeobox protein of Drosophila (Antennapedia), e.g. asdescribed in Chen and Harrison, Biochemical Society Transactions (2007)Volume 35, part 4, p821; Gupta et al. in Advanced Drug Discovery Reviews(2004) Volume 57 9637. Examples of short peptides which have been shownto be efficient at translocation through plasma membranes include the 16amino acid penetratin peptide from Drosophila Antennapedia protein(Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18 aminoacid ‘model amphipathic peptide’ (Oehlke et al (1998) Biochim BiophysActs Volume 1414 p127) and arginine rich regions of the HIV TAT protein.Non peptidic approaches include the use of small molecule mimics orSMOCs that can be easily attached to biomolecules (Okuyama et al (2007)Nature Methods Volume 4 p153). Other chemical strategies to addguanidinium groups to molecules also enhance cell penetration(Elson-Scwab et al (2007) J Biol Chem Volume 282 p13585). Smallmolecular weight molecules such as steroids may be added to themolecular scaffold to enhance uptake into cells.

One class of functional groups which may be attached to peptide ligandsincludes antibodies and binding fragments thereof, such as Fab, Fv orsingle domain fragments. In particular, antibodies which bind toproteins capable of increasing the half-life of the peptide ligand invivo may be used.

In one embodiment, a peptide ligand-effector group according to theinvention has a tβ half-life selected from the group consisting of: 12hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 daysor more, 5 days or more, 6 days or more, 7 days or more, 8 days or more,9 days or more, 10 days or more, 11 days or more, 12 days or more, 13days or more, 14 days or more, 15 days or more or 20 days or more.Advantageously a peptide ligand-effector group or composition accordingto the invention will have a t8 range 12 to 60 hours. In a furtherembodiment, it will have a tβ half-life of a day or more. In a furtherembodiment still, it will be in the range 12 to 26 hours.

In one particular embodiment of the invention, the functional group isselected from a metal chelator, which is suitable for complexing metalradioisotopes of medicinal relevance.

Possible effector groups also include enzymes, for instance such ascarboxypeptidase G2 for use in enzyme/prodrug therapy, where the peptideligand replaces antibodies in ADEPT.

In one particular embodiment of the invention, the functional group isselected from a drug, such as a cytotoxic agent for cancer therapy.Suitable examples include: alkylating agents such as cisplatin andcarboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, ifosfamide; Anti-metabolites including purine analogsazathioprine and mercaptopurine or pyrimidine analogs; plant alkaloidsand terpenoids including vinca alkaloids such as Vincristine,Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and itsderivatives etoposide and teniposide; Taxanes, including paclitaxel,originally known as Taxol; topoisomerase inhibitors includingcamptothecins: irinotecan and topotecan, and type II inhibitorsincluding amsacrine, etoposide, etoposide phosphate, and teniposide.Further agents can include antitumour antibiotics which include theimmunosuppressant dactinomycin (which is used in kidneytransplantations), doxorubicin, epirubicin, bleomycin, calicheamycins,and others.

In one further particular embodiment of the invention, the cytotoxicagent is selected from maytansinoids (such as DM1) or monomethylauristatins (such as MMAE).

DM1 is a cytotoxic agent which is a thiol-containing derivative ofmaytansine and has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent andhas the following structure:

In one embodiment, the cytotoxic agent is linked to the bicyclic peptideby a cleavable bond, such as a disulphide bond or a protease sensitivebond. In a further embodiment, the groups adjacent to the disulphidebond are modified to control the hindrance of the disulphide bond, andby this the rate of cleavage and concomitant release of cytotoxic agent.

Published work established the potential for modifying thesusceptibility of the disulphide bond to reduction by introducing sterichindrance on either side of the disulphide bond (Kellogg et al (2011)Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrancereduces the rate of reduction by intracellular glutathione and alsoextracellular (systemic) reducing agents, consequentially reducing theease by which toxin is released, both inside and outside the cell. Thus,selection of the optimum in disulphide stability in the circulation(which minimises undesirable side effects of the toxin) versus efficientrelease in the intracellular milieu (which maximises the therapeuticeffect) can be achieved by careful selection of the degree of hindranceon either side of the disulphide bond.

The hindrance on either side of the disulphide bond is modulated throughintroducing one or more methyl groups on either the targeting entity(here, the bicyclic peptide) or toxin side of the molecular construct.

In one embodiment, the drug conjugate additionally comprises a linkerbetween said peptide ligand and said cytotoxic agents.

In one embodiment, the cytotoxic agent and linker is selected from anycombinations of those described in WO 2016/067035 (the cytotoxic agentsand linkers thereof are herein incorporated by reference).

In one embodiment the cytotoxic agent is MMAE.

In one embodiment, the linker between said cytotoxic agent and saidbicyclic peptide comprises one or more amino acid residues. Thus, in oneembodiment, the cytotoxic agent is MMAE and the linker is selected from:-Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -Ala-Ala- Asn-,D-Ala-Phe-Lys- or -Glu-Pro-Cit-Gly-hPhe-Tyr-Leu- (SEQ ID NO: 3). In afurther embodiment, the cytotoxic agent is MMAE and the linker isselected from: -Val-Cit-, -Trp-Cit-, -Val-Lys- or -D-Trp-Cit-. In a yetfurther embodiment, the cytotoxic agent is MMAE and the linker is-Val-Cit- or -Val-Lys-. In a still yet further embodiment, the cytotoxicagent is MMAE and the linker is -Val-Cit-.

In an alternative embodiment, the linker between said cytoxic agentcomprises a disulfide bond, such as a cleavable disulfide bond. Thus, ina further embodiment, the cytotoxic agent is DM1 and the linker isselected from: -S-S-, -SS(SO₃H)-, -SS-(Me)-, -(Me)-SS-(Me)-, -SS- (Me₂)-or -SS-(Me)-SO₃H-. In a further embodiment, the cytotoxic agent is DM1and the linker comprises an -S-S- moiety, such as (N-succinimidyl3-(2-pyridyldithio)propionate (SPDB), or an -SS(SO₃H)- moiety, such asSO₃H-SPDB. In a yet further embodiment, the cytotoxic agent is DM1 andthe linker comprises an -S-S- moiety, such as -S-S- or -S-S- (SO₃H)-.

In one embodiment, the cytotoxic agent is DM1 and the drug conjugatecomprises a compound of formula (A):

wherein said bicycle is BCY6099 as defined herein.

In an alternative embodiment, the cytotoxic agent is DM1 and the drugconjugate comprises a compound of formula (B):

wherein said bicycle is BCY6099 as defined herein.

In an alternative embodiment, the cytotoxic agent is DM1 and the drugconjugate comprises a compound of formula (A), wherein said bicycle isselected from BCY6099 as defined herein. This BDC is known herein asBCY6027. Data is presented herein which demonstrates excellentcompetition binding for BCY6027 in the EphA2 competition binding assayas shown in Tables 4 and 8.

In an alternative embodiment, the cytotoxic agent is DM1 and the drugconjugate comprises a compound of formula (B), wherein said bicycle isselected from BCY6099 as defined herein. This BDC is known herein asBCY6028. Data is presented herein which demonstrates excellentcompetition binding for BCY6028 in the EphA2 competition binding assayas shown in Tables 4 and 8.

In a further embodiment, the cytotoxic agent is MMAE or DM1 and the drugconjugate is selected from BCY6136 and BCY6173. Data is presented hereinwhich shows that these two Bicycle Drug Conjugates exhibited nosignificant binding to: closely related human homologs EphA1, EphA3,EphA4, EphA5, EphA6, EphA7 and EphB4; mouse EphA3 and EphA4; and ratEphA3 and EphB1 as shown in Tables 11 and 12.

In a yet further embodiment, the drug conjugate is selected from any oneof: BCY6135, BCY6136, BCY6173, BCY6174 and BCY6175:

In a still yet further embodiment, the drug conjugate is BCY6136. Datais presented herein in Studies 7 and 8 which show that BCY6136 showedsignificant and potent anti-tumor activity in the PC-3 xenograftprostate cancer model (see FIGS. 5 and 6 and Tables 16 to 19). Data isalso provided herein which show that BCY6136 demonstrated potentantitumor activity in the NCI-H1975 xenograft lung cancer (NSCLC) model(see FIG. 8 and Tables 20 to 25). Data is also presented herein inStudies 10 and 11 which show that BCY6136 demonstrated potent anti-tumoreffect in both large and small tumour size LU-01-0251 PDX lung cancer(NSCLC) models (see FIGS. 9 and 10 and Tables 26 to 29) wherein completetumor regression was observed. Data is also presented herein in Study 12which show that BCY6136 demonstrated significant anti-tumor effect inthe LU-01-0046 PDX lung cancer (NSCLC) model (see FIG. 11 and Tables 30and 31) wherein complete tumor regression was observed for BCY6136. Datais also presented herein in Study 13 which show that BCY6136demonstrated dose dependent anti-tumor activity in the LU-01-0046 PDXlung cancer (NSCLC) model (see FIG. 12 and Tables 32 and 33). Data isalso presented herein in Study 14 which show BCY6136 eradicated tumorsin the LU-01-0046 PDX lung cancer (NSCLC) model (see FIGS. 13 to 15 andTables 34 to 37). Data is also presented herein in Studies 15 and 16which demonstrate the effects of BCY6136 in two models which make use ofcell lines with low/negligible EphA2 expression (namely Lu-01-0412 andLu-01-0486). This data is shown in FIGS. 23 and 24 and Tables 38 to 41and demonstrate that BCY6136 had no effect upon tumor regression ineither cell line but BCYs BCY8245 and BCY8781, which bind to a targethighly expressed in the Lu-01-0412 cell line, completely eradicated thetumour. Data is presented herein in Study 17 which show that BCY6136demonstrated potent antitumor activity in the MDA-MB-231 xenograftbreast cancer model (see FIG. 18 and Tables 42 to 45). Data is alsopresented herein in Study 18 which demonstrates the effects of BCY6136in a breast cancer model which makes use of a cell line withlow/negligible EphA2 expression (namely EMT6). This data is shown inFIG. 19 and Tables 46 and 47 and demonstrates that BCY6136 had no effectupon tumor regression in this cell line. Data is also presented hereinin Study 19 which show that BCY6136 demonstrated significant antitumoractivity in the NCI-N87 xenograft gastric cancer model (see FIG. 20 andTables 48 and 49). Data is also presented herein in Study 20 which showthat BCY6136 demonstrated significant antitumor activity in the SK-OV-3xenograft ovarian cancer model (see FIG. 21 and Tables 50 and 51)compared with the ADC MEDI-547 which demonstrated moderate antitumouractivity. Data is also presented herein in Study 21 which show thatBCY6136 demonstrated significant antitumor activity in the OE-21xenograft oesophageal cancer model (see FIG. 22 and Tables 52 and 53).Data is also presented herein in Study 22 which show that BCY6136demonstrated dose-dependent antitumor activity in the MOLP-8 xenograftmultiple myeloma model (see FIG. 23 ). Data is also presented herein inStudy 23 which show that BCY6136 demonstrated potent antitumor activityin the HT-1080 xenograft fibrosarcoma model (see FIGS. 24 to 28 andTables 56 and 57).

Synthesis

The peptides of the present invention may be manufactured syntheticallyby standard techniques followed by reaction with a molecular scaffold invitro. When this is performed, standard chemistry may be used. Thisenables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. Such methods could beaccomplished using conventional chemistry such as that disclosed inTimmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. In one embodiment, thesesteps are carried out on the end product polypeptide/conjugate made bychemical synthesis.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus or within the loops using orthogonallyprotected lysines (and analogues) using standard solid phase or solutionphase chemistry. Standard (bio)conjugation techniques may be used tointroduce an activated or activatable N- or C-terminus. Alternativelyadditions may be made by fragment condensation or native chemicalligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteinsby Native Chemical Ligation. Science 266:776-779), or by enzymes, forexample using subtiligase as described in (Chang et al Proc Natl AcadSci U S

A. 1994 Dec 20; 91(26):12544-8 or in Hikari et al Bioorganic & MedicinalChemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages 6000-6003).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptide to dissociate from each otheronce within the reducing environment of the cell. In this case, themolecular scaffold could be added during the chemical synthesis of thefirst peptide so as to react with the three cysteine groups; a furthercysteine or thiol could then be appended to the N or C-terminus of thefirst peptide, so that this cysteine or thiol only reacted with a freecysteine or thiol of the second peptide, forming a disulfide-linkedbicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of twobicyclic and bispecific macrocycles, potentially creating atetraspecific molecule.

Furthermore, addition of other functional groups or effector groups maybe accomplished in the same manner, using appropriate chemistry,coupling at the N- or C-termini or via side chains. In one embodiment,the coupling is conducted in such a manner that it does not block theactivity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand or a drugconjugate as defined herein in combination with one or morepharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand/or buffered media. Parenteral vehicles include sodium chloridesolution, Ringers dextrose, dextrose and sodium chloride and lactatedRingers. Suitable physiologically-acceptable adjuvants, if necessary tokeep a polypeptide complex in suspension, may be chosen from thickenerssuch as carboxymethylcellulose, polyvinylpyrrolidone, gelatin andalginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringers dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum andimmunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the proteinligands of the present invention, or even combinations of selectedpolypeptides according to the present invention having differentspecificities, such as polypeptides selected using different targetligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, the peptide ligands of the invention canbe administered to any patient in accordance with standard techniques.The administration can be by any appropriate mode, includingparenterally, intravenously, intramuscularly, intraperitoneally,transdermally, via the pulmonary route, or also, appropriately, bydirect infusion with a catheter. Preferably, the pharmaceuticalcompositions according to the invention will be administered byinhalation. The dosage and frequency of administration will depend onthe age, sex and condition of the patient, concurrent administration ofother drugs, counterindications and other parameters to be taken intoaccount by the clinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the peptide ligandsdescribed herein may be used extracorporeally or in vitro selectively tokill, deplete or otherwise effectively remove a target cell populationfrom a heterogeneous collection of cells. Blood from a mammal may becombined extracorporeally with the selected peptide ligands whereby theundesired cells are killed or otherwise removed from the blood forreturn to the mammal in accordance with standard techniques.

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as EphA2binding agents.

Eph receptor tyrosine kinases (Ephs) belong to a large group of receptortyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosineresidues. Ephs and their membrane bound ephrin ligands (ephrins) controlcell positioning and tissue organization (Poliakov et al.

(2004) Dev Cell 7, 465-80). Functional and biochemical Eph responsesoccur at higher ligand oligomerization states (Stein et al. (1998) GenesDev 12, 667-678).

Among other patterning functions, various Ephs and ephrins have beenshown to play a role in vascular development. Knockout of EphB4 andephrin-B2 results in a lack of the ability to remodel capillary bedsinto blood vessels (Poliakov et al., supra) and embryonic lethality.Persistent expression of some Eph receptors and ephrins has also beenobserved in newly-formed, adult micro-vessels (Brantley-Sieders et al.(2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

The de-regulated re-emergence of some ephrins and their receptors inadults also has been observed to contribute to tumor invasion,metastasis and neo-angiogenesis (Nakamoto et al. (2002) Microsc Res Tech59, 58-67; Brantley-Sieders et al., supra). Furthermore, some Eph familymembers have been found to be over-expressed on tumor cells from avariety of human tumors (Brantley-Sieders et al., supra); Marme (2002)Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humansis encoded by the EPHA2 gene.

EphA2 is upregulated in multiple cancers in man, often correlating withdisease progression, metastasis and poor prognosis e.g.: breast(Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010)Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6,e24426), lung (Brannan et al (2009) Cancer Prey Res (Phila) 2,1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al(2013) J Thorac Oncol. 8, 301-308), gastric

(Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2009) Dig DisSci 54, 2410-2417), pancreatic (Mudali et al (2006) Clin Exp Metastasis23, 357-365), prostate (Walker-Daniels et al (1999) Prostate 41,275-280), liver (Yang et al (2009) Hepatol Res. 39, 1169-1177) andglioblastoma (Wykosky et al (2005) Mol Cancer Res. 3, 541-551; Li et al(2010) Tumour Biol. 31, 477-488).

The full role of EphA2 in cancer progression is still not definedalthough there is evidence for interaction at numerous stages of cancerprogression including tumour cell growth, survival, invasion andangiogenesis. Downregulation of EphA2 expression suppresses tumourcancer cell propagation (Binda et al (2012) Cancer Cell 22, 765-780),whilst EphA2 blockade inhibits VEGF induced cell migration (Hess et al(2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng etal (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40)and metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19,1884-1886).

An antibody drug conjugate to EphA2 has been shown to significantlydiminish tumour growth in rat and mouse xenograft models (Jackson et al(2008) Cancer Research 68, 9367-9374) and a similar approach has beentried in man although treatment had to be discontinued for treatmentrelated adverse events (Annunziata et al (2013) Invest New drugs 31,77-84).

Polypeptide ligands selected according to the method of the presentinvention may be employed in in vivo therapeutic and prophylacticapplications, in vitro and in vivo diagnostic applications, in vitroassay and reagent applications, and the like. Ligands having selectedlevels of specificity are useful in applications which involve testingin non-human animals, where cross-reactivity is desirable, or indiagnostic applications, where cross-reactivity with homologues orparalogues needs to be carefully controlled. In some applications, suchas vaccine applications, the ability to elicit an immune response topredetermined ranges of antigens can be exploited to tailor a vaccine tospecific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity arepreferred for administration to a mammal, and 98 to 99% or morehomogeneity is most preferred for pharmaceutical uses, especially whenthe mammal is a human. Once purified, partially or to homogeneity asdesired, the selected polypeptides may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

According to a further aspect of the invention, there is provided apeptide ligand or a drug conjugate as defined herein, for use inpreventing, suppressing or treating a disease or disorder characterisedby overexpression of EphA2 in diseased tissue (such as a tumour).

According to a further aspect of the invention, there is provided amethod of preventing, suppressing or treating a disease or disordercharacterised by overexpression of EphA2 in diseased tissue (such as atumour), which comprises administering to a patient in need thereof aneffector group and drug conjugate of the peptide ligand as definedherein.

In one embodiment, the EphA2 is mammalian EphA2. In a furtherembodiment, the mammalian EphA2 is human EphA2.

In one embodiment, the disease or disorder characterised byoverexpression of EphA2 in diseased tissue is selected from cancer.

Examples of cancers (and their benign counterparts) which may be treated(or inhibited) include, but are not limited to tumours of epithelialorigin (adenomas and carcinomas of various types includingadenocarcinomas, squamous carcinomas, transitional cell carcinomas andother carcinomas) such as carcinomas of the bladder and urinary tract,breast, gastrointestinal tract (including the esophagus, stomach(gastric), small intestine, colon, rectum and anus), liver(hepatocellular carcinoma), gall bladder and biliary system, exocrinepancreas, kidney, lung (for example adenocarcinomas, small cell lungcarcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomasand mesotheliomas), head and neck (for example cancers of the tongue,buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands,nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum,vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (forexample thyroid follicular carcinoma), adrenal, prostate, skin andadnexae (for example melanoma, basal cell carcinoma, squamous cellcarcinoma, keratoacanthoma, dysplastic naevus); haematologicalmalignancies (i.e. leukemias, lymphomas) and premalignant haematologicaldisorders and disorders of borderline malignancy includinghaematological malignancies and related conditions of lymphoid lineage(for example acute lymphocytic leukemia [ALL], chronic lymphocyticleukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma[DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma,T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas,Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy ofuncertain significance, plasmacytoma, multiple myeloma, andpost-transplant lymphoproliferative disorders), and haematologicalmalignancies and related conditions of myeloid lineage (for exampleacute myelogenousleukemia [AML], chronic myelogenousleukemia [CML],chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome,myeloproliferative disorders such as polycythaemia vera, essentialthrombocythaemia and primary myelofibrosis, myeloproliferative syndrome,myelodysplastic syndrome, and promyelocyticleukemia); tumours ofmesenchymal origin, for example sarcomas of soft tissue, bone orcartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas,rhabdomyosarcomas,leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi'ssarcoma, Ewing's sarcoma, synovial sarcomas, epithelioid sarcomas,gastrointestinal stromal tumours, benign and malignant histiocytomas,and dermatofibrosarcomaprotuberans; tumours of the central or peripheralnervous system (for example astrocytomas, gliomas and glioblastomas,meningiomas, ependymomas, pineal tumours and schwannomas); endocrinetumours (for example pituitary tumours, adrenal tumours, islet celltumours, parathyroid tumours, carcinoid tumours and medullary carcinomaof the thyroid); ocular and adnexal tumours (for exampleretinoblastoma); germ cell and trophoblastic tumours (for exampleteratomas, seminomas, dysgerminomas, hydatidiform moles andchoriocarcinomas); and paediatric and embryonal tumours (for examplemedulloblastoma, neuroblastoma, Wilms tumour, and primitiveneuroectodermal tumours); or syndromes, congenital or otherwise, whichleave the patient susceptible to malignancy (for example XerodermaPigmentosum).

In a further embodiment, the cancer is selected from: breast cancer,lung cancer, gastric cancer, pancreatic cancer, prostate cancer, livercancer, glioblastoma and angiogenesis.

In a further embodiment, the cancer is selected from: prostate cancer,lung cancer (such as non-small cell lung carcinomas (NSCLC)), breastcancer (such as triple negative breast cancer), gastric cancer, ovariancancer, oesophageal cancer, multiple myeloma and fibrosarcoma.

In a yet further embodiment, the cancer is prostate cancer. Data ispresented herein in Studies 7 and 8 which show that BCY6136 showedsignificant and potent anti-tumor activity in the PC-3 xenograftprostate cancer model (see FIGS. 5 and 6 and Tables 16 to 19).

In a yet further embodiment, the drug conjugate is useful forpreventing, suppressing or treating solid tumours such as fibrosarcomasand breast, and non-small cell lung carcinomas.

In a yet further embodiment, the cancer is selected from lung cancer,such as non-small cell lung carcinomas (NSCLC). Data is presented hereinin Study 9 which show that BCY6136 demonstrated potent antitumoractivity in the NCI-H1975 xenograft lung cancer (NSCLC) model (see FIG.8 and Tables 20 to 25). Data is also presented herein in Studies 10 and11 which show that BCY6136 demonstrated potent anti-tumor effect in bothlarge and small tumour size LU-01-0251 PDX lung cancer (NSCLC) models(see FIGS. 9 and 10 and Tables 26 to 29) wherein complete tumorregression was observed. Data is also presented herein in Study 12 whichshow that BCY6136 demonstrated significant anti-tumor effect in theLU-01-0046 PDX lung cancer (NSCLC) model (see FIG. 11 and Tables 30 and31) wherein complete tumor regression was observed for BCY6136. Data isalso presented herein in Study 13 which show that BCY6136 demonstrateddose dependent anti-tumor activity in the LU-01-0046 PDX lung cancer(NSCLC) model (see FIG. 12 and Tables 32 and 33). Data is also presentedherein in Study 14 which show that BCY6173 demonstrated antitumoractivity and BCY6136 and BCY6175 eradicated tumors in the LU-01-0046 PDXlung cancer (NSCLC) model (see FIGS. 13 to 15 and Tables 34 to 37). Datais also presented herein in Studies 15 and 16 which demonstrate theeffects of BCY6136 in two models which make use of cell lines withlow/negligible EphA2 expression (namely Lu-01-0412 and Lu-01-0486). Thisdata is shown in FIGS. 23 and 24 and Tables 38 to 41 and demonstratethat BCY6136 had no effect upon tumor regression in either cell line butBCYs BCY8245 and BCY8781, which bind to a target highly expressed in theLu-01-0412 cell line, completely eradicated the tumour. In a furtherembodiment, the cancer is breast cancer. In a yet further embodiment,the breast cancer is triple negative breast cancer. Data is presentedherein in Study 17 which show that BCY6136 demonstrated potent antitumoractivity in the MDA-MB-231 xenograft breast cancer model (see FIG. 18and Tables 42 to 45). Data is also presented herein in Study 18 whichdemonstrates the effects of BCY6136 in a breast cancer model which makesuse of a cell line with low/negligible EphA2 expression (namely EMT6).This data is shown in FIG. 19 and Tables 46 and 47 and demonstrates thatBCY6136 had no effect upon tumor regression in this cell line. In analternative embodiment, the breast cancer is Herceptin resistant breastcancer. Without being bound by theory, EphA2 is believed to beimplicated in the resistance to Herceptin, therefore, an EphA2-targetingentity has potential utility in patients who have failed to respond toHerceptin.

In a further embodiment, the cancer is gastric cancer. Data is presentedherein in Study 19 which show that BCY6136 demonstrated significantantitumor activity in the NCI-N87 xenograft gastric cancer model (seeFIG. 20 and Tables 48 and 49).

In a further embodiment, the cancer is ovarian cancer. Data is presentedherein in Study 20 which show that BCY6136 demonstrated significantantitumor activity in the SK-OV-3 xenograft ovarian cancer model (seeFIG. 21 and Tables 50 and 51) compared with the ADC MEDI-547 whichdemonstrated moderate antitumour activity.

In a further embodiment, the cancer is oesophageal cancer. Data ispresented herein in Study 21 which show that BCY6136 demonstratedsignificant antitumor activity in the OE-21 xenograft oesophageal cancermodel (see FIG. 22 and Tables 52 and 53).

In a further embodiment, the cancer is multiple myeloma. Data ispresented herein in Study 22 which show that BCY6136 demonstrateddose-dependent antitumor activity in the MOLP-8 xenograft multiplemyeloma model (see FIG. 23 ).

In a further embodiment, the cancer is fibrosarcoma. Data is presentedherein in Study 23 which show that BCY6173, BCY6135, BCY6174 and BCY6175demonstrated dose dependent antitumor activity and BCY6136 demonstratedpotent antitumor activity in the HT-1080 xenograft fibrosarcoma model(see FIGS. 24 to 28 and Tables 56 and 57).

References herein to the term “prevention” involves administration ofthe protective composition prior to the induction of the disease.“Suppression” refers to administration of the composition after aninductive event, but prior to the clinical appearance of the disease.“Treatment” involves administration of the protective composition afterdisease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels.

Furthermore, data is presented herein in Study 3 which demonstrates anassociation between copy number variation (CNV) and gene expression forEphA2 from multiple tumor types. Thus, according to a further aspect ofthe invention, there is provided a method of preventing, suppressing ortreating cancer, which comprises administering to a patient in needthereof an effector group and drug conjugate of the peptide ligand asdefined herein, wherein said patient is identified as having anincreased copy number variation (CNV) of EphA2.

In one embodiment, the cancer is selected from those identified hereinas having increased CNV of EphA2. In a further embodiment, the cancer isbreast cancer.

The invention is further described below with reference to the followingexamples.

Examples

Precursor Precursor Abbreviations Name Name CAS Supplier β-Ala β-AlanineFmoc-β-alanine 35737-10-1 Fluorochem D-Asp D-Aspartic acidFmoc-D-aspartic 112883-39-3 Sigma aldrich acid 4-tert-butyl ester SigmaFI 5(6)-carboxyfluorescein HArg HomoArginine Fmoc-L- 401915-53-5Fluorochem HomoArg(Pbf)-OH HyP Hydroxyproline Fmoc- 122996-47-8 SigmaHydroxyproline(tBu)-OH Sar Sarcosine, such that Fmoc-Sarcosine-OH77128-70-2 Sigma Sar_(x) represents × Sar residues

Materials and Methods

Peptide Synthesis

tides were synthesized by solid phase synthesis. Rink Amide MBHA Resinwas used. To a mixture containing Rink Amide MBHA (0.4-0.45 mmol/g) andFmoc-Cys(Trt)-OH (3.0 eq) was added DMF, then DIC (3 eq) and HOAt (3 eq)were added and mixed for 1 hour. 20% piperidine in DMF was used fordeblocking. Each subsequent amino acid was coupled with 3 eq usingactivator reagents, DIC (3.0 eq) and HOAT (3.0 eq) in DMF. The reactionwas monitored by ninhydrin color reaction or tetrachlor color reaction.After synthesis completion, the peptide resin was washed with DMF×3,MeOH×3, and then dried under N₂ bubbling overnight. The peptide resinwas then treated with 92.5% TFA/2.5% TIS/2.5%/EDT/2.5%/H₂O for 3 h. Thepeptide was precipitated with cold isopropyl ether and centrifuged (3min at 3000 rpm). The pellet was washed twice with isopropyl ether andthe crude peptide was dried under vacuum for 2 hours and thenlyophilised. The lyophilised powder was dissolved in of ACN/H₂O (50:50),and a solution of 100 mM TATA in ACN was added, followed by ammoniumbicarbonate in H₂O (1M) and the solution mixed for 1 h. Once thecyclisation was complete, the reaction was quenched with 1M aq. Cysteinehydrochloride (10 eq relative to TATA), then mixed and left to stand foran hour. The solution was lyophilised to afford crude product. The crudepeptide was purified by Preparative HPLC and lyophilized to give theproduct

All amino acids, unless noted otherwise, were used in the L-configurations.

Sequence: (β-Ala)-Sar₁₀-(SEQ ID NO: 2)-CONH₂

8.0 g of resin was used to aenerate 2.1 a BCY6099 (99.2% purity; 16.3%yield) as a white solid.

BCY6099 Analytical Data Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA inACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 μm 110A 150 * 4.6 mmInstrument: Agilent 1200 HPLC-BE(1-614) Method: 15-45% B over 20minutes, then 3 min 95% B Retention Time: 11.31 min LCMS (ESI): m/z1061.8 [M + 3H]³⁺, 796.5 [M + 4H]⁴⁺ Peptide mw 3183.68

Preparation of Bicyclic Peptide Druq Conjugates

The general schematic for preparing Bicycle drug conjugates (BDCs) isshown in FIG. 1 and Table A describes the component targeting bicycleand linker/toxin within each BDC.

TABLE A Targetting Bicycle BDC (BCY Number) (BCY Number) Linker/Toxin6135 6099 DM1-SS- 6136 6099 ValCit-MMAE 6173 6099 DM1-SS(SO₃H)- 61746099 ValLys-MMAE 6175 6099 MMAE-D-Ala-Phe-Lys-

BCY6099 (114.1 mg, 35.84 μmol) was used as the bicycle reagent. 22.4 mgCompound BCY6135 (5.30 μmol, 17.74% yield. 95.14% purity) was obtainedas a white solid.

BCY6135 Analytical Data Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA inACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 μm 110A 150 * 4.6 mmInstrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30minutes, then 3 min 95% B Retention Time: 9.81 LCMS (ESI): m/z 1341.5[M + 3H]³⁺, 805.0 [M + 5H]⁵⁺ Peptide mw 4021.08

BCY6099 (71.5 mg, 22.48 μmol) was used as the bicycle reagent. CompoundBCY6136 (40.9 mg, 9.05 μmol, 40.27% yield, 97.42% purity) was obtainedas a white solid.

BCY6136 Analytical Data Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA inACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 μm 110A 150 * 4.6 mmInstrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30minutes, then 3 min 95% B Retention Time: 11.35 min LCMS (ESI): m/z1468.1 [M + 3H]³⁺, 1101.2 [M + 4H]⁴⁺, 881.3 [M + 5H]⁵⁺ Peptide mw 4404.2

BCY6099 (200.15 mg, 62.89 μmol) was used as the bicycle reagent. 57.1 mgcompound BCY6173 (3.40 μmol, 22.79% yield, 95.80% purity) was obtainedas a white solid.

BCY6173 Analytical Data Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA inACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 μm 110A 150 * 4.6 mmInstrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30minutes, then 3 min 95% B Retention Time: 10.30 min LCMS (ESI): m/z1361.9 [M + 3H − H₂O]³⁺, 1021.8 [M + 4H − H₂O]⁴⁺ Peptide mw 4101.15

BCY6099 (389.77 mg, 122.47 μmol, 1.2 eq) was used as the bicyclereagent. Dde-BCY6174 (0.250 g, 55.10 μmol, 53.99% yield) was obtained asa white solid.

LCMS (ESI): m/z 1513.0 [M + 3H]³⁺, 1135.0 [M + 4H]⁴⁺, 908.2 [M + 5H]⁵⁺Molecular weight 4538.38

Dde-BCY6174 (0.250 g, 55.10 μmol, 1.0 eq) was deprotected usinghydrazine according to the general procedure to give BCY6174 (0.1206 g,27.45 μmol, 49.82% yield) as a white solid.

BCY6174 Analytical Data Mobile Phase: A: 0.1% TFA in H₂O B: 0.1% TFA inACN Flow: 1.0 ml/min Column: Gemini-NX C18 5 μm 110A 150 * 4.6 mmInstrument: Agilent 1200 HPLC-BE(1-614) Method: 28-68% B over 30minutes, then 3 min 95% B Retention Time: 9.85 min LCMS (ESI): m/z1458.5 [M + 3H]³⁺, 1094.1 [M + 4H]⁴⁺, 875.4 [M + 5H]⁵⁺ Peptide mw4373.17

General Procedure for Preparation of Compound 10A

To a solution of BCY6099 (195.15 mg, 61.32 μmol, 1.1 eq) in DMA (3 mL)were added DIEA (21.61 mg, 167.23 μmol, 29.13 μL, 3 eq) and compound 9(0.085 g, 55.74 μmol, 1.0 eq). The mixture was stirred at 25° C. for 16hr. LC-MS showed compound 9 was consumed completely and one main peakwith desired m/z was detected. The reaction mixture was concentratedunder reduced pressure to remove solvent to afford a residue (lightyellow oil). The reaction was directly purified by prep-HPLC (neutralcondition). Compound 10A (0.160 g, 34.84 μmol, 62.50% yield) wasobtained as a white solid.

General Procedure for Preparation of BCY6175

To a solution of compound 10A in DCM (4.5 mL) was added TFA (4.5 mL).The mixture was stirred at 0° C. for 30 min. LC-MS showed compound 10Awas consumed completely and one main peak with desired m/z was detected.The reaction mixture was concentrated under reduced pressure to removesolvent to afford a residue, which was purified by prep-HPLC (TFAcondition). Compound BCY6175 (61.40 mg, 13.56 μmol, 31.13% yield) wasobtained as a white solid.

BIOLOGICAL DATA

Study 1: Fluorescence Polarisation Measurements

(a) Direct Binding Assay

Peptides with a fluorescent tag (either fluorescein, SIGMA or AlexaFluor488™, Fisher Scientific) were diluted to 2.5 nM in PBS with 0.01%tween 20 or 50 mM HEPES with 100 mM NaCl and 0.01% tween pH 7.4 (bothreferred to as assay buffer). This was combined with a titration ofprotein in the same assay buffer as the peptide to give 1 nM peptide ina total volume of 25 μL in a black walled and bottomed low bind lowvolume 384 well plates, typically 5 μL assay buffer, 10 μL protein(Table 1) then 10 μL fluorescent peptide. One in two serial dilutionswere used to give 12 different concentrations with top concentrationsranging from 500 nM for known high affinity binders to 10 μM for lowaffinity binders and selectivity assays. Measurements were conducted ona BMG PHERAstar FS equipped with an “FP 485 520 520” optic module whichexcites at 485 nm and detects parallel and perpendicular emission at 520nm. The PHERAstar FS was set at 25° C. with 200 flashes per well and apositioning delay of 0.1 second, with each well measured at 5 to 10minute intervals for 60 minutes. The gain used for analysis wasdetermined for each tracer at the end of the 60 minutes where there wasno protein in the well. Data was analysed using Systat Sigmaplot version12.0. mP values were fit to a user defined quadratic equation togenerate a Kd value: f=ymin+(ymax−ymin)/Lig*((x+Lig+Kd)/2-sqrt((((x+Lig+Kd)/2){circumflex over( )}2)−(Lig*x))). “Lig” was a defined value of the concentration oftracer used.

(b) Competition Binding Assay

Peptides without a fluorescent tag were tested in competition with apeptide with a fluorescent tag and a known Kd (Table 2). ReferenceCompound A has the sequence FI-G-Sar₅-ACPWGPAWCPVNRPGCA (FI-G-Sar₅- (SEQID NO: 4)). Reference Compound B has the sequenceFI-G-Sar₅-ACPWGPFWCPVNRPGCA (FI-G-Sar₅-(SEQ ID NO: 5)). ReferenceCompound C has the sequence FI-G-Sar₅-ADVTCPWGPFWCPVNRPGCA(FI-G-Sar₅-(SEQ ID NO: 6). Each of Reference Compounds A, B and Ccontain a TBMB molecular scaffold.

Peptides were diluted to an appropriate concentration in assay buffer asdescribed in the direct binding assay with a maximum of 5% DMSO, thenserially diluted 1 in 2. Five pL of diluted peptide was added to theplate followed by 10 μL of human or mouse EphA2 (Table 1) at a fixedconcentration which was dependent on the fluorescent peptide used (Table2), then 10 μL fluorescent peptide added. Measurements were conducted asfor the direct binding assay, however the gain was determined prior tothe first measurement. Data analysis was in Systat Sigmaplot version12.0 where the mP values were fit to a user defined cubic equation togenerate a Ki value:

f=ymin+(ymax−ymin)/Lig*(Lig*((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2-3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}3){circumflex over( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2-3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}3){circumflex over( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))).

“Lig”, “KLig” and “Prot” were all defined values relating to:fluorescent peptide concentration, the Kd of the fluorescent peptide andEphA2 concentration respectively.

TABLE 1 Ephrin receptors and source Catalogue Receptor (domain) SpeciesFormat/tag Supplier number EphA1 (Ecto) Human Fc fusion R&D systems7146-A1 EphA2 (Ecto) Human C-terminal R&D systems 3035-A2 polyHis EphA2(Ecto) Human C-terminal In-house N/A polyHis EphA2 (Ecto) Mouse Fcfusion R&D Systems 639-A2 EphA2 (Ecto) Mouse C-terminal Sino Biological50586-M08H polyHis EphA2 (ligand Rat C-terminal In-house N/A binding)polyHis EphA2 (ligand Dog C-terminal In-house N/A binding) polyHis EphA3(Ecto) Human Fc fusion R&D systems 6444-A3 EphA3 (Ecto) Human N-terminalIn-house N/A polyHis EphA3 (Ecto) Rat C-terminal Sino Biological80465-R08H polyHis EphA4 (Ecto) Human Fc fusion R&D systems 6827-A4EphA4 (Ecto) Human C-terminal Sino Biological 11314-H08H polyHis EphA4(Ecto) Rat C-terminal Sino Biological 80123-R08H polyHis EphA6 (Ecto)Human Fc fusion R&D systems 5606-A6 EphA7 (Ecto) Human Fc fusion R&Dsystems 6756-A7 EphB1 (Ecto) Rat Fc fusion R&D systems 1596-B1 EphB4(Ecto) human C-terminal R&D systems 3038-B4 polyHis

TABLE 2 Final concentrations of fluorescent peptide and EphA2 as usedwith Competition Binding Assays Concentration ConcentrationConcentration of of of Fluorescent fluorescent peptide Human EphA2 MouseEphA2 peptide (nM) (nM) (nM) Reference 10 75 Compound A Reference 1 30Compound B Reference 0.8 (human) 1 (mouse) 2.4 50 Compound C

Certain peptide ligands of the invention were tested in the abovementioned assays and the results are shown in Tables 3 and 4:

TABLE 3 Biological Assay Data for Peptide Ligand of the Invention (TATApeptides Competition Binding Assay) Ki, nM ± 95% Cl Human Human BicycleEphA2 EphA2 Compound Fluorescent Peptide, Number Sequence ScaffoldReference Compound C BCY6099 (β-Ala)-Sar₁₀₋ TATA 4.94 ± 1.41 57.6 ±24.86 A(HArg)DC(HyP)LVNPLCLHP(D- Asp)W(HArg)C (SEQ ID NO: 2)

TABLE 4 Biological Assay Data for Peptide Ligands of the Invention (BDCcompetition binding data with TATA Scaffolds) Ki, nM, Human EphA2 BDCFluorescent Peptide, Compound Bicycle General Reference Number precursorFormula Scaffold Compound C BCY6027 BCY6099 Formula (A) TATA 10.23BCY6028 BCY6099 Formula (B) TATA 13.04

Study 2: Fluorescence Polarisation Measurements (Alternative Protocol)

(a) Competition Binding

Peptides without a fluorescent tag were tested in competition with apeptide with a fluorescent tag and a known Kd (Table 9). Five pL ofincreasing (2 fold) concentrations of test compound was added to theplate followed by 10 μL of EphA2 protein (Table 8) at a fixedconcentration which was dependent on the fluorescent peptide used (Table9), then 10 μL fluorescent peptide added. Buffer was assay buffer asabove with DMSO <1%. Measurements were conducted on a BMG PHERAstar FSequipped with an “FP 485 520 520” optic module which excites at 485 nmand detects parallel and perpendicular emission at 520 nm. The PHERAstarFS was set at 25° C. with 200 flashes per well and a positioning delayof 0.1 second, with each well measured at 5 to 10 minute intervals for60 minutes. Alternatively, measurements were done on at similar timeintervals on a Perkin Elmer Envision equipped with FITC FP Dual Mirror,FITC FP 480 excitation filter and FITC FP P-pol 535 and FITC FP S-polemission filters with 30 flashes and a G-Factor of 1.2. Data analysiswas in Systat Sigmaplot version 12.0 or 13.0 where the mP values at 60minutes were fit to a user defined cubic equation to generate a Kivalue:

f=ymin+(ymax-ymin)/Lin(Lin(2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2-3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2-3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}3){circumflex over( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2-3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2-3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}3){circumflex over( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))). “Lig”, “KLig” and “Prot”were all defined values relating to: fluorescent peptide concentration,the Kd of the fluorescent peptide and EphA2 concentration respectively.

TABLE 5 Eph receptors and source Receptor Catalogue (domain) SpeciesFormat/tag Supplier number EphA2 (Ecto) Human C-terminal R&D 3035-A2polyHis systems EphA2 (Ecto) Human C-terminal In-house N/A polyHis EphA2(Ecto) Mouse C-terminal Sino 50586-M08H polyHis Biological EphA2 (ligandRat C-terminal In-house N/A binding) polyHis

TABLE 6 Final concentrations of fluorescent peptide and EphA2 as usedwith competition binding assays Con- Con- Con- Concentration centrationcentration centration Fluorescent of fluorescent of human of mouse ofrat EphA2 peptide peptide (nM) EphA2 (nM) EphA2 (nM) (nM) Reference 0.82.4 or 25 50 or 15 nM 25 Compound C

Certain peptide ligands and bicycle drug conjugates of the inventionwere tested in the above mentioned competition binding assay and theresults are shown in Table 7:

TABLE 7 Competition Binding with Selected Bicyclic Peptides Human KiMouse Ki Bicycle No. (nM) (nM) Rat Ki (nM) BCY6099 2.7 4.5 1.9

The results from the competition binding assay in Table 7 show thatBicycle peptides targeting human EphA2 (BCY6099) bind with high affinityto mouse and rat EphA2. These results show that the peptide of theinvention can be used in in vivo mouse and rat efficacy and toxicologymodels.

TABLE 8 Competition Binding with Selected Bicycle Drug Conjugates (BDCs)Mouse Bicycle Human Ki Rat Ki ID Ki (nM) (nM) (nM) BCY6027 10.2 BCY602813.0 BCY6135 2.4 5.0 2.9 BCY6136 1.9 5.5 3.2 BCY6173 1.7 4.3 2.5 BCY61741.7 3.9 3.0

Table 8 shows that certain Bicycle Drug Conjugates of the inventionexhibit excellent cross reactivity between human, mouse and rodentEphA2. The peptide of the invention can therefore be used in mouse andrat efficacy and toxicology in vivo models.

(b) SPR Measurements

Non-Fc fusion proteins were biotinylated with EZ-Link™Sulfo-NHS-LC-Biotin for 1 hour in 4 mM sodium acetate, 100 mM NaCl, pH5.4 with a 3× molar excess of biotin over protein. The degree oflabelling was determined using a Fluorescence Biotin Quantification Kit(Thermo) after dialysis of the reaction mixture into PBS. For analysisof peptide binding, a Biacore T200 instrument was used utilising aXanTec CMD500D chip. Streptavidin was immobilized on the chip usingstandard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES,0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyldextran surface was activated with a 7 min injection of a 1:1 ratio of0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 Ξl/min. Forcapture of streptavidin, the protein was diluted to 0.2 mg/ml in 10 mMsodium acetate (pH 4.5) and captured by injecting 120 μl onto theactivated chip surface. Residual activated groups were blocked with a 7min injection of 1 M ethanolamine (pH 8.5):HBS-N (1:1). Buffer waschanged to PBS/0.05% Tween 20 and biotinylated EphA2 was captured to alevel of 500-1500 RU using a dilution of protein to 0.2 μM in buffer. Adilution series of the peptides was prepared in this buffer with a finalDMSO concentration of 0.5% with a top peptide concentration was 50 or100 nM and 6 further 2-fold dilutions. The SPR analysis was run at 25°C. at a flow rate of 90 μl/min with 60 seconds association and 900-1200seconds dissociation. Data were corrected for DMSO excluded volumeeffects. All data were double-referenced for blank injections andreference surface using standard processing procedures and dataprocessing and kinetic fitting were performed using Scrubber software,version 2.0c (BioLogic Software). Data were fitted using simple 1:1binding model allowing for mass transport effects where appropriate.

For binding of Bicycle Drug Conjugates a Biacore 3000 instrument wasused. For biotinylated proteins immobilisation levels were 1500 RU andthe top concentration was 100 nM. Otherwise the method was the same asdescribed above using either the CMD500D or a CM5 chip (GE Healthcare).For the Fc-tagged proteins, a CM5 chip was activated as described aboveand then goat anti-human IgG antibody (Thermo-Fisher H10500) was dilutedto 20pg/ml in 10 mM sodium acetate pH5.0 and captured to approximately3000 RU. The surface was then blocked as described above. Subsequentcapture of the Fc-tagged proteins was carried out to obtainapproximately 200-400 RU of the target protein. The proteins used aredescribed below. All proteins were reconstituted as per manufacturer'ssuggested buffers and concentrations and captured using 5-10 μg/mlprotein in PBS/0.05% Tween 20.

TABLE 9 Catalogue Receptor Species Format/tag Supplier number EphA1Human Fc fusion Sino 15789-H02H Biologics EphA2 Human 0.95 mol In houseN/A biotin/monomer EphA2 Mouse Fc fusion R&D 639-A2 Systems EphA2 Rat1.4 mol biotin/ In house N/A monomer EphA3 Human Fc fusion R&D 6444-A3Systems EphA3 Mouse Fc fusion Sino 51122-M02H Biologics EphA3 Rat Fcfusion Sino 80465-R02H Biologics EphA4 Human Fc fusion Sino 11314-H03HBiologics EphA4 Mouse Fc fusion Sino 50575-M02H Biologics EphA4 Rat Fcfusion Sino 80123-R02H Biologics EphA5 Human 3.1 mol R&D 3036-A5biotin/monomer Systems EphA6 Human Fc fusion R&D 5606-A6 Systems EphA7Human Fc fusion R&D 6756-A7 Systems EphB1 Rat Fc fusion R&D 1596-B1Systems EphB4 Human Fc fusion Sino 10235-H02H Biologics

Certain peptide ligands and bicycle drug conjugates of the inventionwere tested in the above mentioned competition binding assay and theresults are shown in Tables 10 to 12:

TABLE 10 SPR Binding Analysis with Selected Bicyclic Peptides andBicycle Drug Conjugates of the Invention Human Mouse Rat Bicycle/BDCK_(D) K_(off) t_(1/2) K_(on) K_(D) K_(off) t_(1/2) K_(on) K_(D) K_(off)t_(1/2) K_(on) No. (nM) (s − 1) (min) (M − 1s − 1) (nM) (s − 1) (min) (M− 1s − 1) (nM) (s − 1) (min) (M − 1s − 1) BCY6136 1.17 1.15E−03 10.09.86E+05 2.53 1.11E−03 10.4 4.37E+05 2.96 9.11E−04 12.6 3.07E+05 BCY61730.73 1.24E−03 9.3 1.69E+06 2.95 1.14E−03 10.1 3.86E+05 1.10 9.60E−0412.0 8.81E+05

Table 10 details binding affinities and kinetic parameters (Koff andKon) for binding of selected Bicycle Drug Conjugates to human EphA2determined using the SPR assay.

TABLE 11 SPR Binding Analysis with Selected Bicycle Drug Conjugates ofthe Invention with Human Eph Homologs BDC No. EphA1 EphA3 EphA4 EphA5EphA6 EphA7 EphB4 BCY6136 no binding @ no binding @ no binding @ nobinding @ no binding @ no binding @ no binding @ 5 μM 5 μM 5 μM 25 μM 20μM 20 μM 20 μM BCY6173 no binding @ no binding @ no binding @ no binding@ no binding @ no binding @ no binding @ 5 μM 5 μM 5 μM 25 μM 20 μM 20μM 20 μM

Table 11 illustrates binding results with four Bicycle Drug Conjugates(BCY6136 and BCY6173) in the SPR assay with closely related human Ephrinhomologs. The results show that compounds of the invention exhibit nosignificant binding to closely related human homologs: EphA1, EphA3,EphA4, EphA5, EphA6, EphA7 and EphB4.

TABLE 12 SPR Binding Analysis with Selected Bicycle Drug Conjugates ofthe Invention with Mouse and Rat Eph Orthologs Mouse BDC No. EphA3 MouseEphA4 Rat EphA3 Rat EphB1 BCY6136 no no binding @ no binding @ nobinding @ binding @ 20 μM 20 μM 20 μM 20 μM BCY6173 no no binding @ nobinding @ no binding @ binding @ 20 μM 20 μM 20 μM 20 μM

The results in Table 12 show that certain Bicycle Drug Conjugates of theinvention (BCY6136 and BCY6173) are also selective for mouse and ratEphA2 and exhibit no significant binding to closely related homologs:mouse EphA3 and EphA4; and rat EphA3 and EphB1.

Studies 3 and 7-23

In each of Studies 3 and 7-23, the following methodology was adopted foreach study:

(a) Materials

(i) Animals and Housing Condition

Animals

-   -   Species: Mus Musculus    -   Strain: Balb/c nude or CB17-SCID    -   Age: 6-8 weeks    -   Body weight: 18-22 g    -   Number of animals: 9-90 mice    -   Animal supplier: Shanghai Lingchang Biotechnology Experimental        Animal Co. Limited

Housing Condition

-   -   The mice were kept in individual ventilation cages at constant        temperature and humidity with 3-5 animals in each cage.        -   Temperature: 20-26° C.        -   Humidity 40-70%.    -   Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.    -   Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.    -   Water: Animals had free access to sterile drinking water.    -   Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.    -   Animal identification: Animals were marked by ear coding.

(ii) Test and Postitive Control Articles

Molecular Storage Number Physical Description Weight Purity ConditionBCY6135 Lyophilised powder 4021 95.14% Stored at −80° C. BCY6136Lyophilised powder 4402.23 97.5-98.6% Stored at −80° C. BCY6173Lyophilised powder 4101.15 95.80% Stored at −80° C. BCY6174 Lyophilisedpowder 4537 99.50% Stored at −80° C. BCY6175 Lyophilised powder 4492.2996.20% Stored at −80° C. BCY8245 Lyophilised powder 4173.85 99.30%Stored at −80° C. BCY8781 Lyophilised powder 4173.83 99.00% Stored at−80° C. ADC Solution (10.47 mg/ml — >99.00%   Stored at −80° C.(MEDI-547)¹ concentration) ¹Full details of MEDI-547 (a fully humanmonoclonal antibody 1C1 (recognizing both human and murine EphA2)conjugated to MMAF via an mc linker) are described in Jackson et al(2008) Cancer Res 68, 9367-74.

(b) Experimental Methods and Procedures

(i) Observations

All the procedures related to animal handling, care and the treatment inthe study were performed according to the guidelines approved by theInstitutional Animal Care and Use Committee (IACUC) of WuXi AppTec,following the guidance of the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC). At the time of routinemonitoring, the animals were daily checked for any effects of tumorgrowth and treatments on normal behavior such as mobility, food andwater consumption (by looking only), body weight gain/loss, eye/hairmatting and any other abnormal effect as stated in the protocol. Deathand observed clinical signs were recorded on the basis of the numbers ofanimals within each subset.

(ii) Tumor Measurements and the Endpoints

The major endpoint was to see if the tumor growth could be delayed ormice could be cured. Tumor volume was measured three times weekly in twodimensions using a caliper, and the volume was expressed in mm³ usingthe formula: V=0.5 a×b² where a and b are the long and short diametersof the tumor, respectively. The tumor size was then used forcalculations of T/C value. The T/C value (in percent) is an indicationof antitumor effectiveness; T and C are the mean volumes of the treatedand control groups, respectively, on a given day.

TGI was calculated for each group using the formula: TGI(%)=[1-(T_(i)-T₀)/(V_(i)-V₀)]×100; T_(i) is the average tumor volume ofa treatment group on a given day, T₀ is the average tumor volume of thetreatment group on the day of treatment start, V_(i) is the averagetumor volume of the vehicle control group on the same day with T_(i) andV₀ is the average tumor volume of the vehicle group on the day oftreatment start.

(iii) Sample Collection

At the end of study the tumors of all groups were collected for FFPE.

(iv) Statistical Analysis

Summary statistics, including mean and the standard error of the mean(SEM), are provided for the tumor volume of each group at each timepoint.

Statistical analysis of difference in tumor volume among the groups wasconducted on the data obtained at the best therapeutic time point afterthe final dose.

A one-way ANOVA was performed to compare tumor volume among groups, andwhen a significant F-statistics (a ratio of treatment variance to theerror variance) was obtained, comparisons between groups were carriedout with Games-Howell test. All data were analyzed using GraphPad Prism5.0. P<0.05 was considered to be statistically significant.

Study 3: Investigation of Association Between Copy Number Variation(CNV) and Gene Expression for EphA2 from Multiple Tumour Types

Methods

1. Select all studies in cBioPortal (http://www.cboportal.org/) andsearch for EPHA2.

-   -   (a) Remove provisional studies.    -   (b) Deselect studies with overlapping samples to prevent sample        bias (based on warning in cBioPortal)- always keep PanCancer        study if this is an option.    -   (c) Studies selected for analysis (Table 13).

TABLE 13 Studies analysed from cBioPortal and units in study Study NameUnits Breast Invasive Carcinoma (TCGA, mRNA Expression BatchNormalized/Merged PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Lung Squamous Cell Carcinoma mRNA Expression BatchNormalized/Merged (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Kidney Renal Papillary Cell Carcinoma mRNA Expression, RSEM(Batch normalized (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2)Kidney Renal Clear Cell Carcinoma mRNA Expression, RSEM (Batchnormalized (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2) ColonAdenocarcinoma (TCGA, RSEM (Batch normalized from Illumina PanCancerAtlas) HiSeq_RNASeqV2) Head and Neck Squamous Cell mRNA Expression, RSEM(Batch normalized Carcinoma (TCGA, PanCancer Atlas) from IlluminaHiSeq_RNASeqV2) Bladder Urothelial Carcinoma (TCGA, RSEM (Batchnormalized from Illumina PanCancer Atlas) HiSeq_RNASeqV2) Uveal Melanoma(TCGA, PanCancer mRNA Expression Batch Normalized/Merged Atlas) fromIllumina HiSeq_RNASeqV2 syn4976369 Lung Adenocarcinoma (TCGA, mRNAExpression, RSEM (Batch normalized PanCancer Atlas) from IlluminaHiSeq_RNASeqV2) Ovarian Serous Cystadenocarcinoma mRNA Expression BatchNormalized/Merged (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Breast Cancer (METABRIC, Nature mRNA expression (microarray)2012 & Nat Commun 2016) Mesothelioma (TCGA, PanCancer mRNA ExpressionBatch Normalized/Merged Atlas) from Illumina HiSeq_RNASeqV2 syn4976369Colorectal Adenocarcinoma (TCGA, RNA Seq RPKM Nature 2012) CervicalSquamous Cell Carcinoma RSEM (Batch normalized from Illumina (TCGA,PanCancer Atlas) HiSeq_RNASeqV2) Sarcoma (TCGA, PanCancer Atlas) mRNAExpression Batch Normalized/Merged from Illumina HiSeq_RNASeqV2syn4976369 Cancer Cell Line Encyclopedia mRNA expression (microarray)(Novartis/Broad, Nature 2012) Rectum Adenocarcinoma (TCGA, mRNAExpression Batch Normalized/Merged PanCancer Atlas) from IlluminaHiSeq_RNASeqV2 syn4976369 Liver Hepatocellular Carcinoma (TCGA, EPHA2:mRNA Expression, RSEM (Batch PanCancer Atlas) normalized from IlluminaHiSeq_RNASeqV2) Stomach Adenocarcinoma (TCGA, mRNA Expression BatchNormalized/Merged PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Uterine Corpus Endometrial Carcinoma mRNA Expression BatchNormalized/Merged (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Skin Cutaneous Melanoma (TCGA, mRNA Expression BatchNormalized/Merged PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Prostate Adenocarcinoma (TCGA, mRNA Expression, RSEM (Batchnormalized PanCancer Atlas) from Illumina HiSeq_RNASeqV2) KidneyChromophobe (TCGA, mRNA Expression, RSEM (Batch normalized PanCancerAtlas) from Illumina HiSeq_RNASeqV2) Pediatric Wilms' Tumor (TARGET,Epha2: mRNA expression (RNA-Seq RPKM) 2018) Pheochromocytoma and mRNAExpression Batch Normalized/Merged Paraganglioma (TCGA, PanCancer fromIllumina HiSeq_RNASeqV2 syn4976369 Atlas) Thyroid Carcinoma (TCGA,PanCancer mRNA Expression Batch Normalized/Merged Atlas) from IlluminaHiSeq_RNASeqV2 syn4976369 Esophageal Adenocarcinoma (TCGA, RSEM (Batchnormalized from Illumina PanCancer Atlas) HiSeq_RNASeqV2)Cholangiocarcinoma (TCGA, RSEM (Batch normalized from Illumina PanCancerAtlas) HiSeq_RNASeqV2) Brain Lower Grade Glioma (TCGA, RSEM (Batchnormalized from Illumina PanCancer Atlas) HiSeq_RNASeqV2) Thymoma (TCGA,PanCancer Atlas) mRNA Expression Batch Normalized/Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Pediatric Acute Lymphoid Leukemia - Epha2:mRNA expression (RNA-Seq RPKM) Phase II (TARGET, 2018) Diffuse LargeB-Cell Lymphoma mRNA Expression, RSEM (Batch normalized (TCGA, PanCancerAtlas) from Illumina HiSeq_RNASeqV2) Glioblastoma Multiforme (TCGA, mRNAExpression, RSEM (Batch normalized PanCancer Atlas) from IlluminaHiSeq_RNASeqV2) Metastatic Prostate Cancer, SU2C/PCF mRNAexpression/capture (RNA Seq RPKM) Dream Team (Robinson et al., Cell2015) Acute Myeloid Leukemia (TCGA, mRNA Expression, RSEM (Batchnormalized PanCancer Atlas) from Illumina HiSeq_RNASeqV2) TesticularGerm Cell Tumors (TCGA, mRNA Expression Batch Normalized/MergedPanCancer Atlas) from Illumina HiSeq_RNASeqV2 syn4976369 AdrenocorticalCarcinoma (TCGA, RSEM (Batch normalized from Illumina PanCancer Atlas)HiSeq_RNASeqV2) Uterine Carcinosarcoma (TCGA, mRNA Expression BatchNormalized/Merged PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Pancreatic Adenocarcinoma (TCGA, mRNA Expression BatchNormalized/Merged PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Prostate Adenocarcinoma (MSKCC, mRNA Expression Cancer Cell2010) Prostate Adenocarcinoma (Fred mRNA expression Hutchinson CRC, NatMed 2016)

2. Export CNV and RNA expression data from cBioPortal.

3. Test if CNVs are statistically significantly associated with changesin mRNA expression for EphA2 (log2 not applied).

-   -   (a) Run non-parametric Kruskal-Wallis test in GraphPad Prism        (7.04) and R/R studio (threshold for significance: p<0.01).        -   (i) GraphPad Prism: set up column table, run non-parametric            test with no matching or pairing and do not assume Gaussian            distribution.        -   (ii) Packages used in R:            -   1. XLConnect            -   2. dplyr            -   3. Kruskal-Wallis Rank Sum Test: Kruskal.test.

4. Adjust for multiple comparisons (include all possible comparisonseven if n=1 within a group) in R/Rstudio using Dunn's test (thresholdfor significance: p<0.025).

-   -   (a) dunn.test with multiple comparison method=“bonferonni”.

Results

The results are shown in Table 14 below. Across 41 publicly availabledatasets compiled in cBioPortal that report both Copy Number Variation(CNV) and mRNA gene expression for EphA2, there are numerous cancertypes where cases have been reported with EphA2 shallow-deletions (<2copies). Although less common, in these same cancer types a subset oftumors harbored EphA2 deep deletions (>1 copy loss or biallelic loss),EphA2 gains (2-3 copies) or EphA2 amplifications (>3 copies).Indications where >33% of tumors had either shallow-deletions or deepdeletions in EphA2 included: kidney chromophobe, cholangiocarcinoma,pheochromocytoma and paraganglioma, lung squamous cancer, breast,rectum, brain lower grade glioma, liver, adrenocortical carcinoma,mesothelioma, esophageal adenocarcinoma and colon cancer. In contrast,there were no studies where >33% of samples had either gains oramplification in EphA2. Taken together these results demonstrate thatdeletions in EphA2 DNA are found across a variety of indications.

Approximately one third of all samples analyzed in the 41 studiesharbored EphA2 CNVs. Based on this high percentage of CNVs acrossstudies, and the high percentage of shallow deletions within specifictumor types, statistical testing was performed to identify possibleassociations between copy number changes and RNA expression. Tumors perindication were allocated to 1 of 5 classes:

-   -   a) Deep deletion;    -   b) Shallow deletion;    -   c) Diploid;    -   d) Gain; or    -   e) Amplification.

Kruskall-Wallis testing was then performed to detect if thedistributions of mRNA expression values per classes differed betweenclasses (P<0.01). For those TCGA data sets with P<0.01 and to identifywhich classes were different to one another post-hoc testing wasperformed by calculating Z-statistics with adjusted P-values calculated(Bonferroni). For simplicity of interpretation pair-wise comparisons vs.diploid per indication were reviewed (although all pair-wise P-valueswere calculated). 19/41 of these studies had a Kruskall-Wallis p-valueof <0.01 demonstrating that copy number is statistically significantlyassociated with RNA expression. Of these 19 studies, 17 of them had aBonferroni adjusted P<0.025 for Diploid vs. Shallow Deletion indicatingan association of decreased EphA2 mRNA expression with decreased EphA2copy number. Only 2 of these 19 studies had a Bonferroni adjustedP<0.025 for Diploid vs. Gain and both were breast cancer studies.Furthermore, one of these breast cancer studies (Breast InvasiveCarcinoma (TCGA, PanCancer Atlas)) had a Bonferroni adjusted P<0.025 forboth Diploid vs. Shallow Deletion and Diploid vs. Gain suggesting thatcopy number alterations may have a strong impact on EphA2 RNA expressionin breast cancer.

The central dogma of genetics suggests that reduced copy number in EphA2lead to reduced RNA and protein expression. Therefore, the observedassociations between copy number loss of EphA2 and reduced mRNAexpression in a variety of tumor types suggest that EphA2 proteinexpression may also be reduced. Similarly, copy number gains of EphA2 inbreast cancer that were associated with increased mRNA expression mayalso suggest increased EphA2 protein expression. Moreover, higher EphA2protein expression (measured by FACS) is associated with increasedefficacy of certain EphA2 bicyclic drug conjugates of the invention(measured by tumor volume) in preclinical in vivo models. Taken togetherif copy number alterations that are associated with mRNA expressionchanges do predict protein expression levels then patients with tumorscontaining copy number deletions of EphA2 may be less likely to respondto EphA2 bicyclic drug conjugates of the invention.

Similarly, if patients with tumor copy number gains in EphA2 (e.g.breast cancer) it is possible that these patients would be more likelyto respond to EphA2 bicyclic drug conjugates of the invention.Therefore, if patients were stratified by EphA2 copy number status, thenthis information could be used to both exclude and select patients fortreatment with EphA2 bicyclic drug conjugates of the invention toincrease efficacy.

TABLE 14 Results of Investigation of Association between Copy NumberVariation (CNV) and gene expression for EphA2 Kruskal-wallis test Numberof samples/group (n = X) Kruskal- Deep Shallow wallis Study name Unitsdeletion deletion Diploid Gain Amplification statstic p-value BreastInvasive mRNA 5 415 511 61 2 80.816 <2.2e−16 Carcinoma Expression (TCGA,Batch PanCancer Normalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2syn4976369 Lung Squamous mRNA 3 207 201 55 0 52.942 1.89E−11 CellCarcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Mergedfrom Illumina HiSeq_RNASeqV2 syn4976369 Kidney Renal mRNA 1 48 224 0 142.161 3.71E−09 Papillary Cell Expression, Carcinoma RSEM (Batch (TCGA,normalized PanCancer from Illumina Atlas) HiSeq_RNASeqV2) Kidney RenalmRNA 0 69 278 5 0 38.342 4.72E−09 Clear Cell Expression, Carcinoma RSEM(Batch (TCGA, normalized PanCancer from Illumina Atlas) HiSeq_RNASeqV2)Colon RSEM (Batch 3 132 245 8 0 35.397 1.00E−07 Adenocarcinomanormalized (TCGA, from Illumina PanCancer HiSeq_RNASeqV2) Atlas) Headand Neck mRNA 3 86 345 54 0 32.72 3.69E−07 Squamous cell Expression,Carcinoma RSEM (Batch (TCGA, normalized PanCancer from Illumina Atlas)HiSeq_RNASeqV2) Bladder RSEM (Batch 0 73 245 80 4 28.906 2.34E−06Urothelial normalized Carcinoma from Illumina (TCGA, HiSeq_RNASeqV2)PanCancer Atlas) Uveal mRNA 0 24 56 0 0 21.051 4.47E−06 MelanomaExpression (TCGA, Batch PanCancer Normalized/ Atlas) Merged fromIllumina HiSeq_RNASeqV2 syn4976369 Lung mRNA 1 115 263 121 3 28.8748.29E−06 Adenocarcinoma Expression, (TCGA, RSEM (Batch PanCancernormalized Atlas) from Illumina HiSeq_RNASeqV2) Ovarian Serous mRNA 0 5978 60 4 25.349 1.31E−05 Cystadenocarcinoma Expression (TCGA, BatchPanCancer Normalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2syn4976369 Breast Cancer mRNA 1 491 1349 25 0 23.875 2.65E−05 (METABRIC,expression Nature 2012 & (microarray) Nat Commun 2016) Mesothelioma mRNA0 29 50 3 0 18.866 8.00E−05 (TCGA, Expression PanCancer Batch Atlas)Normalized/ Merged from Illumina HiSeq_RNASeqV2 syn4976369 ColorectalRNA Seq 0 53 138 2 0 18.847 8.08E−05 Adenocarcinoma RPKM (TCGA, Nature2012) Cervical RSEM (Batch 1 31 167 76 0 19.435 2.22E−04 Squamous Cellnormalized Carcinoma from Illumina (TCGA, HiSeq_RNASeqV2) PanCancerAtlas) Sarcoma mRNA 0 43 113 70 4 19.389 2.27E−04 (TCGA, ExpressionPanCancer Batch Atlas) Normalized/ Merged from Illumina HiSeq_RNASeqV2syn4976369 Cancer Cell Line mRNA 17 279 418 150 13 20.977 0.00032Encyclopedia expression (Novartis/Broad, (microarray) Nature 2012)Rectum mRNA 1 54 78 3 0 18.215 0.0003971 Adenocarcinoma Expression(TCGA, Batch PanCancer Normalized/ Atlas) Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Liver EPHA2: 1 130 194 21 2 15.514 0.003745Hepatocellular mRNA Carcinoma Expression, (TCGA, RSEM (Batch PanCancernormalized Atlas) from Illumina HiSeq_RNASeqV2) Stomach mRNA 2 90 264 447 13.966 0.007404 Adenocarcinoma Expression (TCGA, Batch PanCancerNormalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2 syn4976369Uterine Corpus mRNA 3 61 395 43 5 12.916 0.0117 Endometrial ExpressionCarcinoma Batch (TCGA, Normalized/ PanCancer Merged from Atlas) IlluminaHiSeq_RNASeqV2 syn4976369 Skin Cutaneous mRNA 2 70 216 72 3 12.2420.01564 Melanoma Expression (TCGA, Batch PanCancer Normalized/ Atlas)Merged from Illumina HiSeq_RNASeqV2 syn4976369 Prostate mRNA 0 44 438 41 10.112 0.01764 Adenocarcinoma Expression, (TCGA, RSEM (Batch PanCancernormalized Atlas) from Illumina HiSeq_RNASeqV2) Kidney mRNA 0 52 12 1 07.8781 0.01947 Chromophobe Expression, (TCGA, RSEM (Batch PanCancernormalized Atlas) from Illumina HiSeq_RNASeqV2) Pediatric Wilms' Epha2:0 22 74 5 0 7.4912 0.02362 Tumor mRNA (TARGET, expression 2018) (RNA-SeqRPKM) Pheochromocytoma mRNA 4 96 60 1 0 8.8074 0.03196 and ExpressionParaganglioma Batch (TCGA, Normalized/ PanCancer Merged from Atlas)Illumina HiSeq_RNASeqV2 syn4976369 Thyroid mRNA 0 4 474 2 0 5.1773 0.08Carcinoma Expression (TCGA, Batch PanCancer Normalized/ Atlas) Mergedfrom Illumina HiSeq_RNASeqV2 syn4976369 Esophageal RSEM (Batch 1 64 8332 1 7.6886 0.1037 Adenocarcinoma normalized (TCGA, from IlluminaPanCancer HiSeq_RNASeqV2) Atlas) Cholangiocarcinoma RSEM (Batch 2 27 7 00 4.1691 0.1244 (TCGA, normalized PanCancer from Illumina Atlas)HiSeq_RNASeqV2) Brain Lower RSEM (Batch 0 191 303 13 0 4.0473 0.1322Grade Glioma normalized (TCGA, from Illumina PanCancer HiSeq_RNASeqV2)Atlas) Thymoma mRNA 0 8 110 1 0 4.0322 1.33E−01 (TCGA, ExpressionPanCancer Batch Atlas) Normalized/ Merged from Illumina HiSeq_RNASeqV2syn4976369 Pediatric Acute Epha2: 1 6 70 4 0 5.5309 0.1368 Lymphoid mRNALeukemia- expression Phase II (RNA-Seq (TARGET, RPKM) 2018) DiffuseLarge B- mRNA 0 4 33 0 0 1.744 0.1866 Cell Lymphoma Expression, (TCGA,RSEM (Batch PanCancer normalized Atlas) from Illumina HiSeq_RNASeqV2)Glioblastoma mRNA 0 13 104 28 0 2.9376 0.2302 Multiforme Expression,(TCGA, RSEM (Batch PanCancer normalized Atlas) from IlluminaHiSeq_RNASeqV2) Metastatic mRNA 2 21 87 7 0 4.069 0.254 Prostateexpression/ Cancer, capture (RNA SU2C/PCF Seq RPKM) Dream Team (Robinsonet al., Cell 2015) Acute Myeloid mRNA 0 1 160 4 0 2.4016 0.301 LeukemiaExpression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IlluminaHiSeq_RNASeqV2) Testicular Germ mRNA 1 29 92 22 0 3.3144 0.3456 CellTumors Expression (TCGA, Batch PanCancer Normalized/ Atlas) Merged fromIllumina HiSeq_RNASeqV2 syn4976369 Adrenocortical RSEM (Batch 0 28 47 10 2.0003 0.3678 Carcinoma normalized (TCGA, from Illumina PanCancerHiSeq_RNASeqV2) Atlas) Uterine mRNA 0 16 22 16 2 2.44 0.4862Carcinosarcoma Expression (TCGA, Batch PanCancer Normalized/ Atlas)Merged from Illumina HiSeq_RNASeqV2 syn4976369 Pancreatic mRNA 2 50 1069 1 3.3833 4.96E−01 Adenocarcinoma Expression (TCGA, Batch PanCancerNormalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2 syn4976369Prostate mRNA 0 5 77 3 0 1.3139 0.5184 Adenocarcinoma Expression (MSKCC,Cancer Cell 2010) Prostate mRNA 0 39 84 10 0 0.028351 0.9859Adenocarcinoma expression (Fred Hutchinson CRC, Nat Med 2016) Pairwisecomparison, Z statistic (adjusted p-value), Bonferonni Deep Diploid-Deletion- Shallow Diploid- Amplification- Study name Units Diploiddeletion Gain Diploid Breast Invasive mRNA 0.176118 6.460580 −4.6031800.713978 Carcinoma Expression (1.0000) (0.0000)* (0.0000)* (1.0000)(TCGA, Batch PanCancer Normalized/ Atlas) Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Lung Squamous mRNA −1.584610 6.786501−0.019607 N/A Cell Carcinoma Expression (0.3392) (0.0000)* (1.0000)(TCGA, Batch PanCancer Normalized/ Atlas) Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Kidney Renal mRNA −1.586207 6.097375 N/A1.549107 Papillary Cell Expression, (0.3381) (0.0000)* (0.3641)Carcinoma RSEM (Batch (TCGA, normalized PanCancer from Illumina Atlas)HiSeq_RNASeqV2) Kidney Renal mRNA N/A 6.133219 −0.487059 N/A Clear CellExpression, (0.0000)* (0.9393) Carcinoma RSEM (Batch (TCGA, normalizedPanCancer from Illumina Atlas) HiSeq_RNASeqV2) Colon RSEM (Batch−2.158194 5.670600 0.781046 N/A Adenocarcinoma normalized (0.0927)(0.0000)* (1.0000) (TCGA, from Illumina PanCancer HiSeq_RNASeqV2) Atlas)Head and Neck mRNA −2.444914 4.680789 −1.530670 N/A Squamous cellExpression, (0.0435) (0.0000)* (0.3776) Carcinoma RSEM (Batch (TCGA,normalized PanCancer from Illumina Atlas) HiSeq_RNASeqV2) Bladder RSEM(Batch N/A 5.203251 0.211744 0.581704 Urothelial normalized (0.0000)*(1.0000) (1.0000) Carcinoma from Illumina (TCGA, HiSeq_RNASeqV2)PanCancer Atlas) Uveal mRNA N/A 4.588095 N/A N/A Melanoma Expression(0.0000)* (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Lung mRNA −0.690460 4.280100 −0.6267072.276458 Adenocarcinoma Expression, (1.0000) (0.0001)* (1.0000) (0.1141)(TCGA, RSEM (Batch PanCancer normalized Atlas) from IlluminaHiSeq_RNASeqV2) Ovarian Serous mRNA N/A 4.390097 −0.239249 0.240543Cystadenocarcinoma Expression (0.0000)* (1.0000) (1.0000) (TCGA, BatchPanCancer Normalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2syn4976369 Breast Cancer mRNA 0.568937 2.274564 −4.115288 N/A (METABRIC,expression (1.0000) (0.0688) (0.0001)* Nature 2012 & (microarray) NatCommun 2016) Mesothelioma mRNA N/A 4.319425 0.170478 N/A (TCGA,Expression (0.0000)* (1.0000) PanCancer Batch Atlas) Normalized/ Mergedfrom Illumina HiSeq_RNASeqV2 syn4976369 Colorectal RNA Seq N/A 4.298092−0.338975 N/A Adenocarcinoma RPKM (0.0000)* (1.0000) (TCGA, Nature 2012)Cervical RSEM (Batch −1.618248 3.42960 −1.446339 N/A Squamous Cellnormalized (0.3168) (0.0018)* (0.4442) Carcinoma from Illumina (TCGA,HiSeq_RNASeqV2) PanCancer Atlas) Sarcoma mRNA N/A 3.666949 −0.8524540.953027 (TCGA, Expression (0.0007)* (1.0000) (1.0000) PanCancer BatchAtlas) Normalized/ Merged from Illumina HiSeq_RNASeqV2 syn4976369 CancerCell Line mRNA −2.084879 −3.615935 −2.007004 −0.108880 Encyclopediaexpression (0.1854) (0.0015)* (0.2237) (1.0000) (Novartis/Broad,(microarray) Nature 2012) Rectum mRNA −1.926519 3.877166 1.167400 N/AAdenocarcinoma Expression (0.1621) (0.0003)* (0.7291) (TCGA, BatchPanCancer Normalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2syn4976369 Liver EPHA2: 0.302341 3.697248 −0.336659 0.454454Hepatocellular mRNA (1.0000) (0.0011)* (1.0000) (1.0000) CarcinomaExpression, (TCGA, RSEM (Batch PanCancer normalized Atlas) from IlluminaHiSeq_RNASeqV2) Stomach mRNA −2.072978 1.606072 −1.750466 1.602806Adenocarcinoma Expression (0.1909) (0.5413) (0.4002) (0.5449) (TCGA,Batch PanCancer Normalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2syn4976369 Uterine Corpus mRNA −1.905863 1.039307 −1.597383 2.268798Endometrial Expression (0.2833) (1.0000) (0.5509) (0.1164) CarcinomaBatch (TCGA, Normalized/ PanCancer Merged from Atlas) IlluminaHiSeq_RNASeqV2 syn4976369 Skin Cutaneous mRNA 1.094526 2.674493 0.0959661.692628 Melanoma Expression (1.0000) (0.0374) (1.0000) (0.4526) (TCGA,Batch PanCancer Normalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2syn4976369 Prostate mRNA N/A 2.905502 1.374609 −0.082790 AdenocarcinomaExpression, (0.0110)* (0.5078) (1.0000) (TCGA, RSEM (Batch PanCancernormalized Atlas) from Illumina HiSeq_RNASeqV2) Kidney mRNA N/A 2.4983401.863169 N/A Chromophobe Expression, (0.0187)* (0.0937) (TCGA, RSEM(Batch PanCancer normalized Atlas) from Illumina HiSeq_RNASeqV2)Pediatric Wilms' Epha2: N/A 2.690766 −0.173274 N/A Tumor mRNA (0.0107)*(1.0000) (TARGET, expression 2018) (RNA-Seq RPKM) Pheochromocytoma mRNA−1.411567 2.201344 1.946134 N/A and Expression (0.4742) (0.0831)(0.1549) Paraganglioma Batch (TCGA, Normalized/ PanCancer Merged fromAtlas) Illumina HiSeq_RNASeqV2 syn4976369 Thyroid mRNA N/A 2.2218840.503577 N/A Carcinoma Expression (0.0394) (0.9218) (TCGA, BatchPanCancer Normalized/ Atlas) Merged from Illumina HiSeq_RNASeqV2syn4976369 Esophageal RSEM (Batch −1.462679 0.910990 −1.682311 −0.362298Adenocarcinoma normalized (0.7178) (1.0000) (0.4625) (1.0000) (TCGA,from Illumina PanCancer HiSeq_RNASeqV2) Atlas) Cholangiocarcinoma RSEM(Batch −2.037840 0.972100 N/A N/A (TCGA, normalized (0.0623) (0.4965)PanCancer from Illumina Atlas) HiSeq_RNASeqV2) Brain Lower RSEM (BatchN/A 0.722383 −1.771514 N/A Grade Glioma normalized (0.7051) (0.1147)(TCGA, from Illumina PanCancer HiSeq_RNASeqV2) Atlas) Thymoma mRNA N/A1.982334 0.369115 N/A (TCGA, Expression (0.0712) (1.0000) PanCancerBatch Atlas) Normalized/ Merged from Illumina HiSeq_RNASeqV2 syn4976369Pediatric Acute Epha2: 1.437404 −0.805100 1.607586 N/A Lymphoid mRNA(0.4518) (1.0000) (0.3238) Leukemia- expression Phase II (RNA-Seq(TARGET, RPKM) 2018) Diffuse Large B- mRNA N/A 1.320613 N/A N/A CellLymphoma Expression, (0.0933) (TCGA, RSEM (Batch PanCancer normalizedAtlas) from Illumina HiSeq_RNASeqV2) Glioblastoma mRNA N/A 1.428778−0.716110 N/A Multiforme Expression, (0.2296) (0.7109) (TCGA, RSEM(Batch PanCancer normalized Atlas) from Illumina HiSeq_RNASeqV2)Metastatic mRNA −1.812613 0.992571 0.314089 N/A Prostate expression/(0.2097) (0.9628) (1.0000) Cancer, capture (RNA SU2C/PCF Seq RPKM) DreamTeam (Robinson et al., Cell 2015) Acute Myeloid mRNA N/A −1.539142−0.199532 N/A Leukemia Expression, (0.1857) (1.0000) (TCGA, RSEM (BatchPanCancer normalized Atlas) from Illumina HiSeq_RNASeqV2) TesticularGerm mRNA 0.574846 −0.443110 −1.751161 N/A Cell Tumors Expression(1.0000) (1.0000) (0.2398) (TCGA, Batch PanCancer Normalized/ Atlas)Merged from Illumina HiSeq_RNASeqV2 syn4976369 Adrenocortical RSEM(Batch N/A 1.346397 0.550103 N/A Carcinoma normalized (0.2673) (0.8734)(TCGA, from Illumina PanCancer HiSeq_RNASeqV2) Atlas) Uterine mRNA N/A0.476071 −0.550292 1.215102 Carcinosarcoma Expression (1.0000) (1.0000)(0.6730) (TCGA, Batch PanCancer Normalized/ Atlas) Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Pancreatic mRNA −1.195082 0.159442 −0.6025581.217697 Adenocarcinoma Expression (1.0000) (1.0000) (1.0000) (1.0000)(TCGA, Batch PanCancer Normalized/ Atlas) Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Prostate mRNA N/A −0.406579 −1.089948 N/AAdenocarcinoma Expression (1.0000) (0.4136) (MSKCC, Cancer Cell 2010)Prostate mRNA N/A 0.160404 0.079785 N/A Adenocarcinoma expression(1.0000) (1.0000) (Fred Hutchinson CRC, Nat Med 2016)

Study 4: In Vivo Efficacy of BCY6136 in CDX Xenograft Models

The study evaluated the therapeutic efficacy of BCY6136 in three CancerCell Line Derived (CDX) models: the HT1080 fibrosarcoma line, theMDA-MB-231 triple negative breast cancer line and the NCI-H1975non-small cell lung cancer (NSCLC) line.

(a) Experimental method

Balb/c mice were inoculated subcutaneously with tumour cells at theright flank and drug treatment started when the average the averagetumour volume reached between 150 and 200 mm³. Tumour measurements andstatistical analysis were performed as described above. Tumour bearinganimals were treated once weekly with BCY6136 or vehicle.

(b) Discussion

FIGS. 4-6 show that BCY6136 is effective in breast, lung andfibrosarcoma xenograft models following once weekly dosing.

The HT1080 Fibrosarcoma Model:

In the HT1080 model complete regression of tumour growth was achieved byday 14 following once weekly dosing with BCY6136 on days 0 and 7 at 3and 5 mg/kg (FIG. 2 ). Once weekly dosing with BCY6136 at 2 mg/kg ondays 0 and 7 gave rise to tumour stasis (partial regression) (FIG. 2 ).BCY6136 treatment gave rise to no significant body weight loss (FIG. 2inset) and there were no adverse clinical observations on drug treatedmice throughout the study.

The NCI-H1975 NSCLC Model:

Complete regression of tumour growth in the NCI-H1975 model was observedby around day 28 following 2 and 3 mg/kg once weekly dosing with BCY6136(FIG. 3 ). Following dosing cessation on day 35 no tumour regrowth wasobserved in the 3 mg/kg treated animals from day 35 to day 72 when the 3mg/kg arm measurements ended (FIG. 3 ). Dosing with BCY6136 at 2 mg/kggave rise to complete regression in this model from around day 28.Following dosing cessation on day 35 there was no tumour regrowth untilaround day 51 at the 2 mg/kg dose. At this dose level moderate tumourre-growth was observed from around day 51 until study termination on day77. 1 mg/kg treatment with BCY6136 gave rise to tumour stasis (partialregression) (FIG. 3 ). BCY6136 treatment gave rise to no significantbody weight loss (FIG. 3 inset) and there were no adverse clinicalobservations on drug treated mice throughout the study.

The MDA-MB-231 Breast Model:

Tumour stasis (partial regression) was observed in the MDA-MB231 modelfollowing once weekly dosing at 2 and 3 mg/kg from days 0 to day 45(FIG. 4 ). Some body weight loss (attributed to tumour burden) wasobserved in the 2 mg/kg treated animals (FIG. 4 inset).

These results demonstrate that BCY6136 gives rise to profound tumourgrowth inhibition in mice implanted with fibrosarcoma, breast and lungCDX xenografts following once daily dosing.

Study 5: Safety Studies in the Rat

Six (6) female rats were randomly assigned to 3 groups of 2 rats/groupto determine the toxicity of BCY6136, following administered by IV bolusinjection at 5, 7.5 and 10 mg/kg on days 1 and 8. The study wasterminated on day 15.

No significant effects on coagulation parameters (Prothrombin time(sec), Activated partial thromboplastin time (sec) or Fibroginogenlevels (g/L) were observed on days 2, 12 and 15 (data not shown). Noin-life bleeding events were reported and no evidence of internalbleeding was detected following pathology examination.

Study 6: Safety Studies in the Cynomologous Monkeys

Twenty eight day toxicology studies with BCY6136 we conducted incynomologous monkeys. BCY6136 was dosed at 1.0 and 2.0 mg/kg on days 1,8, 15 and 22. Animals were euthanised and necropsied on day 29 (7 daysafter the final dose).

No significant effects on coagulation parameters relative to baselinewere observed on days 18, 22 and 25 (data not shown) and day 29 (Table15). No in-life bleeding events were reported and no evidence ofinternal bleeding was detected following pathology examination.

TABLE 15 Day 29 coagulation parameters following 1.0 and 2.0 mg/kgBCY6136 dosing to cynomolgus monkeys 1.0 mg/kg × 4 2.0 mg/kg × 4Baseline Day 29 Baseline Day 29 PT(s) 13.4 11.7 9.4 9.7 PT(s) 11 9.211.2 11.0 APTT(s) 18.9 19.4 19.4 20.9 APTT(s) 16.1 15.7 18.7 18.2FIB(g/L) 2.08 2.42 1.86 6.1 FIB(g/L) 2.28 2.35 1.82 3.1

Study 7: In Vivo Efficacy Study of BCY6136 and ADC in Treatment of PC-3Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of PC-3 xenograft.

(b) ExperimentalDesign

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10iv qw 4 BCY6136 3 3 10 iv qw 5 ADC 3 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The PC-3 tumor cells will be maintained in F12K medium supplemented with10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5%CO₂ in air. The tumor cells will be routinely subcultured twice weekly.The cells growing in an exponential growth phase will be harvested andcounted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse will be inoculated subcutaneously at the right flank withPC-3 (10*10⁶) tumor cells for tumor development. The animals will berandomized and treatment will be started when the average tumor volumereaches approximately 150 mm³. The test article administration and theanimal numbers in each group are shown in the following experimentaldesign table.

(iii) Testing Article Formulation Preparation

Con. Test article (mg/ml) Formulation Vehicle — 50 mM Acetate/aceticacid pH 5 10% sucrose BCY6136 0.1 Dilute 90 μl 1 mg/ml BCY6136 stockwith 810 μl vehicle buffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with720 μl vehicle buffer 0.3 Dilute 270 μl 1 mg/ml BCY6136 stock with 630μl vehicle buffer ADC 0.3 Dilute 26 μl 10.47 mg/ml ADC stock with 874 μlADC buffer

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIGS. 5 and 6 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing PC-3xenograft is shown in Table 16.

TABLE 16 Tumor volume trace over time Gr Treatment 0 2 4 7 9 11 14 16 1821 1 Vehicle, 149 ± 9  235 ± 9  377 ± 9  718 ± 30 1126 ± 41  1431 ± 79 1792 ± 69  2070 ± 152 qw 2 BCY6136, 150 ± 11 185 ± 25 228 ± 31 201 ± 17183 ± 23 153 ± 38 137 ± 33 107 ± 32 64 ± 28 45 ± 23 1 mpk, qw 3 BCY6136,149 ± 18 179 ± 28 158 ± 22 137 ± 16 122 ± 15 114 ± 20 101 ± 16  79 ± 2057 ± 19 42 ± 17 2 mpk, qw 4 BCY6136 149 ± 2  155 ± 8  144 ± 16 132 ± 20107 ± 28  94 ± 23  83 ± 22  70 ± 27 38 ± 16 35 ± 17 3 mpk, qw 5 ADC 151± 27 203 ± 10 210 ± 12 189 ± 11 185 ± 16 190 ± 37 158 ± 36 124 ± 35 103± 27  74 ± 14 3 mpk, qw Gr Treatment 23 25 28 30 32 35 37 39 42 1Vehicle, qw 2 BCY6136, 35 ± 18 28 ± 14 37 ± 19 34 ± 17 42 ± 21 42 ± 2343 ± 21 28 ± 14 18 ± 9 1 mpk, qw 3 BCY6136, 21 ± 11 22 ± 12 22 ± 12 24 ±12 33 ± 16 22 ± 11 26 ± 14 22 ± 12 16 ± 9 2 mpk, qw 4 BCY6136 21 ± 10 23± 12 27 ± 14 22 ± 11 24 ± 12 20 ± 11 27 ± 14 12 ± 6  12 ± 6 3 mpk, qw 5ADC 53 ± 16 50 ± 22 46 ± 23 70 ± 35 78 ± 39 53 ± 27 60 ± 30 53 ± 27  40± 22 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis Tumor growth inhibition rate fortest articles in the PC-3 xenograft model was calculated based on tumorvolume measurements at day 16 after the start of treatment.

TABLE 17 Tumor growth inhibition analysis Tumor T/C^(b) TGI P valuecompare Gr Treatment Volume (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw2070 ± 152 — — — 2 BCY6136, 107 ± 32 5.2 102.2 p < 0.001 1 mpk, qw 3BCY6136,  79 ± 20 3.8 103.6 p < 0.001 2 mpk, qw 4 BCY6136,  70 ± 27 3.4104.1 p < 0.001 3 mpk, qw 5 ADC, 124 ± 35 6.0 101.4 p < 0.001 3 mpk, qw^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the PC-3xenograft model was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points are shown in theFIGS. 5 and 6 and Tables 16 and 17.

The mean tumor size of vehicle treated mice reached 2070 mm³ on day 16.BCY6136 at 1 mg/kg, qw (TV=107 mm³, TGI=102.2%, p<0.001), BCY6136 at 2mg/kg, qw (TV=79 mm³, TGI=103.6%, p<0.001) and BCY6136 at 3 mg/kg, qw(TV=70 mm³, TGI=104.1%, p<0.001) showed potent anti-tumor effect. Inthis study, animal body weight was monitored regularly. All micemaintained their body weight well.

Study 8. In vivo efficacy study of BCY6136 in treatment of PC-3xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of PC-3 xenograft in Balb/c nude mice.

(b) Experimental Design

Dose Dosing Group Treatment (mg/kg) N^(a) Route Schedule 1 Vehicle — 4i.v.  qw × 4 weeks 2 BCY6136 0.167 4 i.v.  qw × 4 weeks  3^(b) BCY61360.5 4 i.v.  qw × 4 weeks 4 BCY6136 1.5 4 i.v.  qw × 4 weeks  5^(b)BCY6136 0.5 4 i.v. q2w × 2 weeks  6^(b) BCY6136 1.5 4 i.v. q2w × 2 weeks7 EphA2-ADC 0.33 4 i.v.  qw × 4 weeks 8 EphA2-ADC 1 4 i.v.  qw × 4 weeks9 EphA2-ADC 3 4 i.v.  qw × 4 weeks 10^(c ) Docetaxel 15 4 i.v.  qw × 4weeks ^(a)N, the number of animals in each group. ^(b)After 4 weeks'treatment demonstrated in the experimental design table, the mice ofgroup 3, 5 and 6 were treated with BCY6136 1.5 mg/kg qw from day 52during the monitoring schedule. ^(c)Due to the severe body weight lossof the Docetaxel treated mice after the first dosing, the treatment wassuspended for 2 weeks, then a lower dosage (Docetaxel, 10 mg/kg) wasperformed on day 28. After that, the mice were treated with BCY6136 1.5mg/kg qw from day 42 to day 70.

(c) Experimental Methods and Procedures

(i) Cell Culture

The tumor cells were maintained in F-12K medium supplemented with 10%heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂in air. The tumor cells were routinely subcultured twice weekly. Thecells growing in an exponential growth phase were harvested and countedfor tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with PC-3tumor cells (10×10⁶) in 0.2 ml of PBS for tumor development. 52 animalswere randomized when the average tumor volume reached 454 mm³. The testarticle administration and the animal numbers in each group were shownin the experimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article Purity (mg/ml) Formulation Vehicle — — 25 mMHistidine pH 7 10% sucrose BCY6136 98.6% — 50 mM Acetate 10% sucrose pH5 1 Dissolve 2.70 mg BCY6136 in 2.662 ml Acetate buffer 0.3 Dilute 300μl 1 mg/ml BCY6136 stock with 700 μl Acetate buffer¹ 0.15 Dilute 600 μl0.3 mg/ml BCY6136 stock with 600 μl Acetate buffer 0.05 Dilute 200 μl0.3 mg/ml BCY6136 stock with 1000 μl Acetate buffer 0.0167 Dilute 66.7μl 0.3 mg/ml BCY6136 stock with 1133.3 μl Acetate buffer EphA2- — — 25mM Histidine pH 5.5 ADC 0.033 Dilute 9.3 μl 4.24 mg/ml EphA2-ADC stockwith 1191 μl His buffer 0.1 Dilute 28 μl 4.24 mg/ml EphA2-ADC stock with1172 μl His buffer 0.3 Dilute 84.9 μl 4.24 mg/ml EphA2-ADC stock with1115 μl His buffer Docetaxel — 10 Mix 0.5 ml 20 mg Docetaxel with 1.5 mlbuffer 1.5 Dilute 180 μl 10 mg/ml Docetaxel stock with 1020 μl salinebuffer ¹50 mM Acetate 10% sucrose pH 5 3. 25 mM Histidine pH 5.5

(c) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 7 .

(ii) Tumor Volume Trace

Mean tumor volume over time in male Balb/c nude mice bearing PC-3xenograft is shown in Table 18.

TABLE 18 Tumor volume trace over time (Day 0 to day 20) Days after thestart of treatment Gr. Treatment 0 2 4 6 8 10 1 Vehicle, qw 456 ± 25 648± 50 880 ± 23 1022 ± 29  1178 ± 118 1327 ± 133 2 BCY6136 450 ± 33 631 ±55 695 ± 78 739 ± 39 850 ± 68 904 ± 73 0.167 mpk, qw 3 BCY6136 451 ± 47622 ± 96 519 ± 70 460 ± 55 398 ± 50 329 ± 38 0.5 mpk, qw 4 BCY6136 458 ±49 587 ± 63 494 ± 54 363 ± 32 283 ± 32 237 ± 24 1.5 mpk, qw 5 BCY6136454 ± 37 643 ± 25 531 ± 37 458 ± 33 411 ± 32 382 ± 49 0.5 mpk, q2w 6BCY6136 452 ± 42 590 ± 75 457 ± 49 375 ± 44 328 ± 47 242 ± 63 1.5 mpk,q2w 1.5 mpk, qw 7 EphA2-ADC 457 ± 43 636 ± 57 712 ± 70 792 ± 78 870 ± 87900 ± 58 0.33 mpk, qw 8 EphA2-ADC 450 ± 49 617 ± 48 673 ± 50 721 ± 61782 ± 78 755 ± 67 1 mpk, qw 9 EphA2-ADC 452 ± 60 593 ± 98 643 ± 141  593± 106  433 ± 103 290 ± 81 3 mpk, qw 10  Docetaxel 453 ± 62 584 ± 72 632± 56 636 ± 48 568 ± 50 408 ± 31 15 mpk, qw Days after the start oftreatment Gr. Treatment 13 15 17 20 1 Vehicle, qw 1631 ± 93  1868 ± 90 2052 ± 139 2364 ± 102 2 BCY6136 975 ± 47 1089 ± 74  1124 ± 92  1188 ±111 0.167 mpk, qw 3 BCY6136 260 ± 33 249 ± 33 231 ± 38 234 ± 42 0.5 mpk,qw 4 BCY6136 192 ± 13 164 ± 16 155 ± 20 131 ± 19 1.5 mpk, qw 5 BCY6136430 ± 88  522 ± 124  560 ± 129  530 ± 147 0.5 mpk, q2w 6 BCY6136 206 ±61 197 ± 62 182 ± 55 128 ± 36 1.5 mpk, q2w 1.5 mpk, qw 7 EphA2-ADC 1049± 66  1242 ± 123 1443 ± 129 1637 ± 181 0.33 mpk, qw 8 EphA2-ADC 840 ± 93913 ± 91  978 ± 100  981 ± 100 1 mpk, qw 9 EphA2-ADC 268 ± 64 232 ± 60225 ± 66 184 ± 62 3 mpk, qw 10  Docetaxel 374 ± 26 388 ± 36 361 ± 25 419± 31 15 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the PC-3 xenograftmodel was calculated based on tumor volume measurements at day 20 afterthe start of the treatment.

TABLE 19 Tumor growth inhibition analysis P value Tumor compared VolumeT/C^(b) TGI with Gr Treatment (mm³)^(a) (%) (%) vehicle 1 Vehicle, qw2364 ± 102 — — — 2 BCY6136, 0.167 mpk, qw 1188 ± 111 50.2 61.4 p < 0.0013 BCY6136, 0.5 mpk, qw 234 ± 42 9.9 111.4 p < 0.001 4 BCY6136, 1.5 mpk,qw 131 ± 19 5.5 117.2 p < 0.001 5 BCY6136, 0.5 mpk, q2w  530 ± 147 22.496.0 p < 0.001 6 BCY6136, 1.5 mpk, q2w 128 ± 36 5.4 117.0 p < 0.001 7EphA2-ADC, 0.33 mpk, 1637 ± 181 69.2 38.1 p < 0.001 qw 8 EphA2-ADC, 1mpk, qw  981 ± 100 41.5 72.2 p < 0.001 9 EphA2-ADC, 3 mpk, qw 184 ± 627.8 114.0 p < 0.001 10 Docetaxel, 15 mpk, qw 419 ± 31 17.7 101.8 p <0.001 ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated bydividing the group average tumor volume for the treated group by thegroup average tumor volume for the control group (T/C).

(d) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the PC-3xenograft model was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points are shown in theFIG. 7 and Tables 18 and 19.

The mean tumor size of vehicle treated mice reached 2364 mm³ on day 20.BCY6136 at 0.167 mg/kg, qw (TV=1188 mm³, TGI=61.4%, p<0.001), 0.5 mg/kg,q2w (TV=530 mm³, TGI=96.0%, p<0.001), 0.5 mg/kg, qw (TV=234 mm³,TGI=111.4%, p<0.001) and 1.5 mg/kg, qw (TV=131 mm³, TGI=117.2%, p<0.001)produced significant anti-tumor activity in dose or dose-frequencydependent manner on day 20. BCY6136 at 1.5 mg/kg, q2w (TV=128 mm³,TGI=117.0%, p<0.001) produced comparable anti-tumor activity withBCY6136 1.5 mg/kg qw. Among them, the mice treated with BCY6136, 0.5mg/kg qw or BCY6136, 0.5 mg/kg q2w showed obvious tumor relapse afterceasing the treatment, further treatment with BCY6136, 1.5 mg/kg qw fromday 52 worked well on the tumor regression. The mice treated withBCY6136, 1.5 mg/kg q2w also showed tumor relapse after ceasing thetreatment, but further dosing didn't work on complete tumor regression.The mice treated with BCY6136, 1.5 mpk qw didn't show any tumor relapseuntil day 48.

EphA2-ADC at 0.33 mg/kg, qw (TV=1637 mm³, TGI=38.1%, p<0.001), 1 mg/kg,qw (TV=981 mm³, TGI=72.2%, p<0.001) and 3 mg/kg, qw (TV=184 mm³,TGI=114.0%, p<0.001) produced significant anti-tumor activity in dosedependent manner on day 20. The mice treated with EphA2-ADC, 3 mg/kg qwdidn't show any tumor relapse until day 59. Docetaxel at 15 mg/kg, qw(TV=419 mm³, TGI=101.8%, p<0.001) produced significant anti-tumoractivity but caused severe animal body weight loss. After ceasing thetreatment, the mice showed obvious tumor relapse. The treatment withBCY6136, 1.5 mg/kg qw from day 42 worked well on tumor regression ofthese mice.

Study 9. In Vivo Efficacy Test of BCY6136 in Treatment of NCI-H1975Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of NCI-H1975 xenograft model in Balb/cnude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10iv qw 4 BCY6136 3 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The cells growing in an exponential growth phase were harvested andcounted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withNCI-H1975 tumor cells (10×10{circumflex over ( )}6) in 0.2 ml of PBS fortumor development. 36 animals were randomized when the average tumorvolume reached 149 mm³. The test article administration and the animalnumbers in each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Dose Treatment (mg/ml) Formulation Vehicle 50 mM Acetate, 10% sucrose pH= 5 BCY6136 1 Dissolve 3.79 mg BCY6136 in 3.695 ml formulation buffer0.3 Dilute 270 μl 1 mg/ml BCY6136 with 630 μl formulation buffer 0.2Dilute 180 μl 1 mg/ml BCY6136 with 720 μl formulation buffer 0.1 Dilute90 μl 1 mg/ml BCY6136 with 810 μl formulation buffer

(iv) Sample Collection

On PG-D44, we fixed the tumors of Group 2 for FFPE.

At the end of study, we the tumors of Group 3 for FFPE.

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth are shown in FIG. 8 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing NCI-H1975xenograft is shown in Table 20 to 24.

TABLE 20 Tumor volume trace (PG-D 0~PG-D 17) Days after the start oftreatment Gr. Treatment 0 2 4 7 9 11 14 17 1 Vehicle, qw 148 ± 4 195 ±11 297 ± 33 466 ± 64  732 ± 107 1028 ± 192 1278 ± 252 1543 ± 298 2BCY6136, 150 ± 6 178 ± 20 232 ± 49 336 ± 43 400 ± 24 407 ± 42  299 ± 113 261 ± 127 1 mpk, qw 3 BCY6136,  150 ± 14 181 ± 26 237 ± 27 277 ± 36 297± 37 306 ± 55 256 ± 53 218 ± 49 2 mpk, qw 4 BCY6136, 148 ± 9 168 ± 10231 ± 6  365 ± 16 390 ± 13 423 ± 42 319 ± 26 228 ± 16 3 mpk, qw

TABLE 21 Tumor volume trace (PG-D 18~PG-D 35) Days after the start oftreatment Gr. Treatment 18 21 23 25 28 30 33 35 1 Vehicle, qw 1864 ± 3952371 ± 470  — — — — — — 2 BCY6136,  215 ± 113 205 ± 117 197 ± 113 200 ±105 202 ± 112 202 ± 117  230 ± 142 241 ± 127 1 mpk, qw 3 BCY6136, 149 ±31 99 ± 30 69 ± 22 42 ± 13 30 ± 10 16 ± 8  20 ± 9 4 ± 2 2 mpk, qw 4BCY6136, 149 ± 17 94 ± 30 50 ± 15 41 ± 21 21 ± 8  6 ± 6 10 ± 6 3 ± 1 3mpk, qw

TABLE 22 Tumor volume trace (PG-D 37~PG-D 53) Days after the start oftreatment Gr. Treatment 37 39 42 44 46 49 51 53 2 BCY6136, 277 ± 149 294± 159 351 ± 188 — — — — — 1 mpk, qw 3 BCY6136, 7 ± 4 2 ± 1 1 ± 0 3 ± 1 2± 1 3 ± 2 6 ± 3 14 ± 10 2 mpk, qw 4 BCY6136, 3 ± 3 2 ± 1 1 ± 0 0 ± 0 0 ±0 0 ± 0 1 ± 0 1 ± 0 3 mpk, qw

TABLE 23 Tumor volume trace (PG-D 56~PG-D 74) Days after the start oftreatment Gr. Treatment 56 58 60 63 65 67 70 72 74 3 BCY6136, 16 ± 11 27± 18 34 ± 23 45 ± 31 63 ± 40 71 ± 47 95 ± 70 111 ± 73 122 ± 75 2 mpk, qw4 BCY6136, 1 ± 0 1 ± 0 1 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0  0 ± 0 — 3 mpk, qw

TABLE 24 Tumor volume trace (PG-D 77~PG-D 98) Days after the start oftreatment Gr. Treatment 77 81 84 88 91 95 98 3 BCY6136, 208 ± 112 337 ±123 501 ± 172 626 ± 182 856 ± 245 1035 ± 169 1266 ± 39 2 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the NCI-H1975 xenograftmodel was calculated based on tumor volume measurements at day 21 afterthe start of treatment.

TABLE 25 Tumor growth inhibition analysis Tumor Gr Treatment Volume(mm³)^(a) T/C^(b) (%) TGI (%) P value 1 Vehicle, qw 2371 ± 470  — — — 2BCY6136, 205 ± 117 8.6 97.5 p < 0.001 1 mpk, qw 3 BCY6136, 99 ± 30 4.2102.3 p < 0.001 2 mpk, qw 4 BCY6136, 94 ± 30 4.0 102.4 p < 0.001 3 mpk,qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the NCI-H1975xenograft model was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points are shown in theFIG. 8 and Tables 20 to 25.

The mean tumor size of vehicle treated mice reached 2371 mm³ on day 21.BCY6136 at 1 mg/kg (TV=205 mm³, TGI=97.5%, p<0.001), 2 mg/kg (TV=99 mm³,TGI=102.3%, p<0.001) and 3 mg/kg (TV=94 mm³, TGI=102.4%, p<0.001)produced potent antitumor activity. BCY6136 at 2 mg/kg and 3 mg/kgeradicated the tumors or regressed the tumor to small size. Thetreatments was suspended from day 35, and the tumors in 3 mg/kg groupdidn't show obvious re-growth in following 5-6 weeks monitoring, howevertumors in 2 mg/kg group showed obvious regrowth and didn't showsignificant tumor inhibition when resuming the dosing. In this study,mice maintained the bodyweight well.

Study 10. In Vivo Efficacy Study of BCY6136 in the LU-01-0251 PDX Modelin Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in the LU-01-0251 PDX model in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY6136 5 1 10 iv qw 3 BCY6136 5 2 10iv qw 4 BCY6136 5 3 10 iv qw 5 ADC 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withLU-01-0251 of tumor fragment (˜30 mm³) for tumor development. Thetreatment was started when the average tumor volume reached 174 mm³ forefficacy study. The test article administration and the animal number ineach group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10%sucrose pH 5 BCY6136 0.3 Dissolve 6.11 mg BCY6136 in 20 ml Acetatebuffer¹ 0.2 Dilute 940 μl 0.3 mg/ml BCY6136 stock with 470 μl Acetatebuffer 0.1 Dilute 470 μl 0.3 mg/ml BCY6136 stock with 940 μl Acetatebuffer ADC 0.3 Dilute 43 μl 10.47 mg/ml ADC stock with 1457 μl ADCbuffer² ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5 ²ADC buffer: 20mM Histidine pH 5.5

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 9 .

(ii) Tumor Volume Trace

Mean tumor volume on day 28 after the start of treatment in femaleBalb/c nude mice bearing LU-01-0251 xenograft is shown in Table 26.

TABLE 26 Tumor volume trace over time Group 2 Group 3 Group 4 Group 5Group 1 BCY6136, BCY6136, BCY6136, ADC, Day Vehicle 1 mpk, qw 2 mpk, qw3 mpk, qw 3 mpk, qw 0 174 ± 17 175 ± 15  174 ± 17  175 ± 14 174 ± 16 3264 ± 33 230 ± 29  205 ± 21  187 ± 19 227 ± 12 7 403 ± 68 281 ± 55  154± 21  118 ± 13 239 ± 42 10 562 ± 83 370 ± 104 111 ± 19   72 ± 12 241 ±46 14  777 ± 163 362 ± 104 62 ± 17 30 ± 5 191 ± 47 17 1021 ± 246 437 ±136 46 ± 13 17 ± 3 139 ± 39 21 1472 ± 342 526 ± 167 30 ± 18  4 ± 3 101 ±31 24 1790 ± 417 491 ± 132 32 ± 24  1 ± 1  70 ± 23 28 2208 ± 512 499 ±128 32 ± 30  0 ± 0  39 ± 14

(iii) Tumor Growth Inhibition Analysis p Tumor growth inhibition ratefor BCY6136 and ADC in the LU-01-0251 PDX model was calculated based ontumor volume measurements at day 28 after the start of the treatment.

TABLE 27 Tumor growth inhibition analysis Tumor Group Treatment Volume(mm³)^(a) T/C^(b) (%) TGI (%) P value 1 Vehicle, qw 2208 ± 512  — — — 2BCY6136, 1 mpk, qw 499 ± 128 22.6 84.0 p < 0.001 3 BCY6136, 2 mpk, qw 32± 30 1.4 107.0 p < 0.001 4 BCY6136, 3 mpk, qw 0 ± 0 0.0 108.6 p < 0.0015 ADC, 3 mpk, qw 39 ± 14 1.8 106.6 p < 0.001 ^(a)Mean ± SEM; ^(b)TumorGrowth Inhibition is calculated by dividing the group average tumorvolume for the treated group by the group average tumor volume for thecontrol group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 and ADC in LU-01-0251PDX model was evaluated. The measured body weight and tumor volume ofall treatment groups at various time points are shown in the FIG. 9 andTables 26 and 27.

In this study, the mean tumor volume of vehicle treated mice reached2208 mm³ on day 28 after the start of treatment. BCY6136 at 1 mg/kg, qw(TV=499 mm³, TGI=84.0%, p<0.001), 2 mg/kg, qw (TV=32 mm³, TGI=107.0%,p<0.001) and 3 mg/kg, qw (TV=0 mm³, TGI=108.6%, p<0.001) produceddose-dependent anti-tumor activity. ADC at 3 mg/kg, qw (TV=39 mm³,TGI=106.6%, p<0.001) showed significant anti-tumor activity.

Study 11: In Vivo Efficacy Study of BCY6136 in the LU-01-0251 PDX Modelin Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in the LU-01-0251 PDX model in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1  Vehicle 5 — 10 iv Qw * 21 2  BCY6136 5 1 10 iv Qw * 28 3^(a)BCY6136 5 2 10 iv Qw * 70 4^(b) BCY6136 5 3 10 iv Qw * 56 5^(c) ADC 5 310 iv Qw * 70 ^(a)The dosing schedule was kept from day 0 to day 70 forall the mice of this group, then the mouse 3-2 and mouse 3-4 werefurther dosed with BCY6136 3 mg/kg qw from day 77 while the treatment ofthe other 3 mice was suspended. The dosing schedule was kept from day 0to day 56 for all the mice of this group. ^(b)The dosing schedule waskept from day 0 to day 70 for all the mice of this group.

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withLU-01-0251 of tumor fragment (˜-30 mm³) for tumor development. Thetreatment was started when the average tumor volume reached 960 mm³ forefficacy study. The test article administration and the animal number ineach group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 25 mM Histidine 10%sucrose pH 7 BCY6136 0.3 0.3 mg/ml BCY6136 was prepared as in Study 10hereinbefore 0.2 Dilute 940 μl 0.3 mg/ml BCY6136 stock with 470 μlHis-buffer¹ 0.1 Dilute 470 μl 0.3 mg/ml BCY6136 stock with 940 μlHis-buffer ADC 0.3 Dilute 43 μl 10.47 mg/ml ADC stock with 1457 μlADC-buffer² ¹His-buffer: 25 mM Histidine 10% sucrose pH 7 ²ADC-buffer:20 mM Histidine pH 5.5

(iii) Sample Collection

Tumor of mouse #3-2 was collected for FFPE on Day 94. Tumors of mice#5-2 and 5-3 were collected and embed into 1 FFPE block on Day 140.

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 10 .

(ii) Tumor Volume Trace

Mean tumor volume on day 0 to day 28 after the start of treatment infemale Balb/c nude mice bearing LU-01-0251 xenograft is shown in Table28.

TABLE 28 Tumor volume trace over time Group 2 Group 3 Group 4 Group 5Group 1 BCY6136, BCY6136, BCY6136, ADC, Day Vehicle 1 mpk, qw 2 mpk, qw3 mpk, qw 3 mpk, qw 0  962 ± 102 963 ± 97 962 ± 137 960 ± 103  959 ± 1243 1176 ± 108 1003 ± 121 973 ± 105 989 ± 128 1043 ± 158 7 1351 ± 142 1056± 151 873 ± 125 890 ± 98 1100 ± 156 10 1591 ± 179 1122 ± 139 722 ± 157674 ± 96 1172 ± 188 14 1951 ± 225 1417 ± 191 503 ± 151 342 ± 64 1228 ±174 17 2301 ± 344 1672 ± 262 398 ± 160 216 ± 43 1143 ± 186 21 1794 ± 328307 ± 169  94 ± 26  996 ± 187 24 1867 ± 408 261 ± 168  62 ± 14  867 ±178 28 2120 ± 483 217 ± 167  45 ± 16  713 ± 178

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 and ADC in the LU-01-0251 PDXmodel was calculated based on tumor volume measurements at day 17 afterthe start of the treatment.

TABLE 29 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GroupTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 2301 ± 344 — — — 2BCY6136, 1 mpk, qw 1672 ± 262 72.7 47.0 p > 0.05  3 BCY6136, 2 mpk, qw 398 ± 160 17.3 142.1 p < 0.001 4 BCY6136, 3 mpk, qw 216 ± 43 9.4 155.6p < 0.001 5 ADC, 3 mpk, qw 1143 ± 186 49.7 86.3 p < 0.01  ^(a)Mean ±SEM; ^(b)Tumor Growth Inhibition is calculated by dividing the groupaverage tumor volume for the treated group by the group average tumorvolume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 and ADC in LU-01-0251PDX model was evaluated. The measured body weight and tumor volume ofall treatment groups at various time points are shown in the FIG. 10 andTables 28 and 29.

In this study, the treatment was started when the average tumor volumereached 960 mm³. On day 17 after the start of treatment, the mean tumorvolume of vehicle treated mice reached 2301 mm³. BCY6136 at 1 mg/kg qw(TV=1672 mm³, TGI=47.0%, p>0.05) didn't show obvious antitumor activity;BCY6136 at 2 mg/kg qw (TV=398 mm³, TGI=142.1%, p<0.001) and 3 mg/kg qw(TV=216 mm³, TGI=155.6%, p<0.001) produced dose-dependent anti-tumoractivity on day 17.

After 70 days' treatment with BCY6136 at 2 mg/kg qw, 3 in 5 of thesemice showed complete tumor regression, the other 2 mice showed obvioustumor relapse from day 42 to day 77. Then further treatment with BCY61363 mg/kg qw was performed to the two relapse tumors from day 7, one oftumor showed obvious tumor regress while another one showed resistanceto the treatment.

After 56 days' treatment with BCY6136 at 3 mg/kg qw, all the mice ofthis group showed complete tumor regression.

ADC at 3 mg/kg qw (TV=1143 mm³, TGI=86.3%, p<0.01) showed obviousanti-tumor activity on day 17, after another 53 day' treatment, thesemice showed further but not complete tumor regression.

In this study, there were some mice showed sudden bodyweight loss, thismay have the relationship with the long term feeding of theimmune-deficiency mice.

Study 12: In Vivo Efficacy Study of BCY6136 in the LU-01-0046 NSCLC PDXModel in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in large LU-01-0046 PDX tumors in Balb/c nude mice.

(b) Experimental Design

Dose Dosing Dosing Group Treatment n (mg/kg) Volume (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY6136 5 1 10 iv qw 3 BCY6136 5 3 10iv qw 4 ADC 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withLU-01-0046 of tumor fragment (˜30 mm³) for tumor development. Thetreatment was started when the average tumor volume reaches 1039 mm³.The test article administration and the animal numbers in each group areshown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10%sucrose pH 5 BCY6136 0.1 Dilute 150 μl 1 mg/ml BCY6136 stock with 1350μl Acetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6136 stock with 1050 μlAcetate buffer ADC 0.3 Dilute 43 μl 10.47 mg/ml ADC stock solution into1457 μl with buffer² ¹Acetate buffer: 50 mM Acetate 10% sucrose pH5²Dissolve 0.419 g His. hydrochloride in 100 ml water, use 1M HCl adjustPH to 5.5

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 11 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearingLU-01-0046 is shown in Table 30.

TABLE 30 Tumor volume trace over time (BCYs Section) Days after thestart of treatment Group Treatment 0 4 8 11 15 18 22 1 Vehicle, qw 1044± 115 1762 ± 178 2404 ± 262 — — — — 3 mpk, qw 2 BCY6136, 1037 ± 130 1163± 146 1927 ± 283 2483 ± 530 — — — 1 mpk, qw 3 BCY6136, 1036 ± 100  784 ±146  548 ± 107  362 ± 110 325 ± 122 275 ± 152 233 ± 187 3 mpk, qw 4 ADC,1033 ± 114 1155 ± 230 2200 ± 505 — — — — 3 mpk, qw 3 mpk, qw Note: thetumor volume trace didn't show after the day 22 for the group 2 and 4.

(iii) Tumor Growth Inhibition Analysis Tumor growth inhibition rate fortest articles in the LU-01-0046 PDX model was calculated based on tumorvolume measurements at day 22 and day 28 respectively for the twosection studies after the start of the treatment.

TABLE 31 Tumor growth inhibition analysis (BCYs section on day 22) TumorGroup Treatment Volume T/C^(b) (%) TGI (%) P value 1 Vehicle, 6186 ±596* — — — qw 2 BCY6136, 4564 ± 981* 73.8 31.4 p > 0.05  1 mpk, qw 3BCY6136,  233 ± 187 3.8 115.6 p < 0.001 3 mpk, qw 4 ADC, 5446 ± 1250*88.0 14.2 p > 0.05  3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor GrowthInhibition is calculated by dividing the average tumor volume of thetreated group by the average tumor volume of the control group (T/C).*Some groups was terminated before day 22, and the tumor size wascalculated by exponential growth equation acquisition as below: Vehiclegroup: Y = 995.4 × exp (0.1134 × X). BCY6136, 1 mpk group: Y = 855.0 ×exp (0.0974 × X). ADC, 3 mpk group: Y = 757.4 × exp (0.1312 × X).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in largeLU-01-0046 tumors was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points are shown in theFIG. 11 and Tables 30 and 31.

In this study, the mean tumor size of vehicle treated mice wascalculated as 6186 mm³ on day 22. BCY6136 at 1 mg/kg and ADC at 3mg/kgdidn't show obvious anti-tumor activity when starting treatment fromtumor size of 1000mm³.

BCY6136 (TV=233 mm³, TGI=115.6%, p<0.001) at 3 mg/kg producedsignificant anti-tumor antitumor activity. In particular, BCY6136eradicated 2/5 and 4/5 tumors completely.

Study 13: In Vivo Efficacy of BCY6136 in Balb/c Nude Mice BearingLU-01-0046 NSCLC PDX Model

(a) Study Objective

The objective of the research was to evaluate the in vivo therapeuticefficacy of BCY6136 in Balb/c nude mice bearing LU-01-0046 NSCLC PDXmodel.

(b) Experimental Design

Dose Dosing Group Treatment n (mg/kg) Route Schedule 1 Vehicle 5 — i.v.qw * 2 w 2 BCY6136 5 1 i.v. qw * 3 w 3 BCY6136 5 2 i.v. qw * 4 w 4BCY6136 5 3 i.v. qw * 4 w 5 ADC 5 3 i.v. qw * 3 w 6 ADC 5 5 i.v. qw * 3w Note: Groups were terminated when average tumor volume reached over2000 mm³ and tumors were harvested for FFPE: Group 1 on PG-D14, group 5on PG-D18, group 2 & 6 on PG-D21 and group 3 & 4 on PG-D31.

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with certainkind of tumor fragment (˜30 mm³) for tumor development. The treatmentswere started when the average tumor volume reached approximately 198mm³. The test article administration and the animal numbers in eachgroup are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Dose Gr Compounds (mg/kg) Con. (mg/ml) Formulation 1 Vehicle — — 50 mMAcetate, 10% Sucrose pH 5 (without DMSO) 2 BCY6136 1 0.1 Dissolve 10.93mg BCY6136 in 10.766 ml vehicle, ultrasonic simply to make the 1 mg/mlBCY6136 stock solution Dilute 150 μl 1 mg/ml BCY6136 stock solution with1350 μl vehicle 3 BCY6136 2 0.2 Dilute 300 μl 1 mg/ml BCY6136 stocksolution with 1200 μl vehicle 4 BCY6136 3 0.3 Dilute 450 μl 1 mg/mlBCY6136 stock solution with 1050 μl vehicle Buffer 2: Dissolve 0.419 gHis. hydrochloride in 100 ml water, use 1M HCl adjust pH to 5.5 5 ADC 30.3 Dilute 43 μl 10.47 mg/ml ADC stock solution with 1457 μl with buffer2 6 ADC 5 0.5 Dilute 71.6 μl 10.47 mg/ml ADC stock solution with1428.4μl with buffer 2 Note: The dosing formulation frequently is freshprepared timely.

(iii) Sample Collection

Groups were terminated when average tumor volume reached over 2000 mm³and tumors were harvested for FFPE after the last measurement: Group 1on PG-D14, group 5 on PG-D18, group 2 & 6 on PG-D21 and group 3 & 4 onPG-D31.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 12 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearingLU-01-0046 NSCLC PDX model is shown in Table 32.

TABLE 32 Tumor volume trace over time (mm³) Gr 1 2 3 4 5 6 VehicleBCY6136 BCY6136 BCY6136 ADC ADC Treatment qw 1 mpk, qw 2 mpk, qw 3 mpk,qw 3 mpk, qw 5 mpk, qw 0 201 ± 37 198 ± 39 201 ± 40 200 ± 46  195 ± 28195 ± 40 3 441 ± 82 310 ± 59 283 ± 77 155 ± 40  418 ± 99 389 ± 68 7  927± 171 547 ± 88  423 ± 132 74 ± 19  643 ± 159  596 ± 116 10 1546 ± 377 747 ± 121  321 ± 108 31 ± 8   938 ± 230  882 ± 134 14 2307 ± 594 1058 ±140 264 ± 95 26 ± 11 1475 ± 466 1215 ± 193 17 — 1390 ± 205 127 ± 41 26 ±13 2281 ± 556 1576 ± 228 21 — 2138 ± 301 118 ± 34 64 ± 42 — 2049 ± 24224 — — 101 ± 40 99 ± 63 — — 28 — —  255 ± 140 276 ± 176 — — 31 — —  582± 346 477 ± 283 — —

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in Balb/c nude micebearing LU-01-0046 PDX model was calculated based on tumor volumemeasured on PG-D14.

TABLE 33 Tumor growth inhibition analysis P value Tumor compared Volumewith Gr Treatment (mm³)^(a) T/C (%)^(b) TGI (%)^(c) vehicle 1 Vehicle2307 ± 594 — — — qw 2 BCY6136 1058 ± 140 45.9 59.1 p < 0.05  1 mpk, qw 3BCY6136 264 ± 95 11.4 97.0 p < 0.001 2 mpk, qw 4 BCY6136  26 ± 11 1.1108.3 p < 0.001 3 mpk, qw 5 ADC 1475 ± 466 63.9 39.2 p > 0.05  3 mpk, qw6 ADC 1215 ± 193 52.7 51.6 p > 0.05  5 mpk, qw ^(a)Mean ± SEM. ^(b)TumorGrowth Inhibition was calculated by dividing the group average tumorvolume for the treated group by the group average tumor volume for thecontrol group (T/C). ^(c)TGI was calculated for each group using theformula: TGI (%) = [1 − (T_(i) − T₀)/(V_(i) − V₀)] × 100

(e) Results Summary and Discussion

In the present study, the therapeutic efficacy of test articles in theLU-01-0046 PDX model was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points were shown in theFIG. 12 and Tables 32 and 33.

The mean tumor size of vehicle treated mice reached 2307 mm³ on PG-D14.BCY6136 at 1 mg/kg (TV=1058 mm³, TGI=59.1%, p<0.05), at 2 mg/kg (TV=264mm³, TGI=97.0%, p<0.001) and at 3 mg/kg (TV=26 mm³, TGI=108.3%, p<0.001)produced dose-dependent antitumor activity. ADC at 3 mg/kg and 5 mg/kgdid not show obvious antitumor activity (p>0.05).

In this study, all of the group's animals maintained the body weightwell.

Study 14: In Vivo Efficacy Study of BCY6136, BCY6173 and BCY6175 in theLU 0046 NSCLC PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in the LU-01-0046 NSCLC PDX model in Balb/cnude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule Part 1 1 Vehicle 5 — 10 iv qw 2 BCY6136 5 ½ 10 iv qw 3 BCY61365 3 10 iv qw Part 2 4 Vehicle 5 — 10 iv qw 5 BCY6173 5 1 10 iv qw 6BCY6173 5 3 10 iv qw 7 BCY6175 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withLU-01-0046 of tumor fragment (˜30 mm³) for tumor development. Thetreatment was started when the average tumor volume reaches 200 mm³ forpart 1 study and 192 mm³ for part 2 study. The test articleadministration and the animal numbers in each group are shown in theexperimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10%sucrose pH 5 BCY6136 0.1 Dilute 150 μl 1 mg/ml BCY6136 stock with 1350μl Acetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6136 stock with 1050 μlAcetate buffer BCY6173 0.1 Dissolve 3.65 mg BCY6173 in 3.5 ml Acetatebuffer to make 1 mg/ml stock. Dilute 150 μl 1 mg/ml BCY6173 with 1350 μlAcetate buffer 0.3 Dilute 450 μl 1 mg/ml BCY6173 stock with 1050 μlAcetate buffer BCY6175 0.3 Dissolve 3.02 mg BCY6175 in 2.9 ml Acetatebuffer to make 1 mg/ml stock. Dilute 450 μl 1 mg/ml BCY6175 with 1050 μlAcetate buffer ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5

(d) Ressults

Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIGS. 13 to 15 .

(ii) Tumor Volume Trace

Mean tumor volume on day 21 after the start of treatment in femaleBalb/c nude mice bearing LU-01-0046 is shown in Tables 34 and 35.

TABLE 34 Tumor volume trace over time (Part 1) Days after the start oftreatment Gr Treatment 0 3 6 10 14 17 21 1 Vehicle, qw 202 ± 26 328 ± 48536 ± 68 953 ± 107 1386 ± 97  1833 ± 132  2551 ± 242 2 BCY6136, 200 ± 33293 ± 56 426 ± 91 682 ± 151  964 ± 194 976 ± 258 1285 ± 234 1 mpk, qw 3BCY6136, 201 ± 33 194 ± 31 135 ± 27 52 ± 18 13 ± 9 4 ± 4  0 ± 0 3 mpk,qw

TABLE 35 Tumor volume trace over time (Part 2) Days after the start oftreatment Gr Treatment 0 3 7 10 14 17 21 4 Vehicle, qw 192 ± 30 311 ± 83562 ± 146 830 ± 230 1320 ± 444 1652 ± 528 2342 ± 651 5 BCY6173, 191 ± 33318 ± 58 553 ± 88  817 ± 165 1314 ± 276 1546 ± 276 2151 ± 262 1 mpk, qw6 BCY6173, 192 ± 37 259 ± 51 400 ± 53  455 ± 28  636 ± 92  646 ± 138 890 ± 260 3 mpk, qw 7 BCY6175, 192 ± 42 186 ± 57 92 ± 38 19 ± 11  0 ± 0 0 ± 0  0 ± 0 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the LU-01-0046 PDXmodel was calculated based on tumor volume measurements at day 21 afterthe start of the treatment.

TABLE 36 Tumor growth inhibition analysis (Part 1) Tumor Group TreatmentVolume T/C^(b) (%) TGI (%) P value 1 Vehicle, 2551 ± 242 — — — qw 2BCY6136, 1285 ± 234 50.4 53.9 p < 0.001 1 mpk, qw 3 BCY6136,  0 ± 0 0.0108.5 p < 0.001 3 mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition iscalculated by dividing the group average tumor volume for the treatedgroup by the group average tumor volume for the control group (T/C).

TABLE 37 Tumor growth inhibition analysis (Part 2) Tumor Group TreatmentVolume T/C^(b) (%) TGI (%) P value 4 Vehicle, 2342 ± 651 — — — qw 5BCY6173, 2151 ± 262 91.8 8.9 p > 0.05  1 mpk, qw 6 BCY6173,  890 ± 26038.0 67.5 p < 0.05  3 mpk, qw 7 BCY6175,  0 ± 0 0.0 108.9 p < 0.001 3mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated bydividing the group average tumor volume for the treated group by thegroup average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in theLU-01-0046 PDX model was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points are shown in theFIGS. 13 to 15 and Tables 34 to 37.

In part 1 study, the mean tumor size of vehicle treated mice reached2551 mm³ on day 21 after the start of treatment.

BCY6136 at 1/2 mg/kg, qw (TV=1285 mm³, TGI=53.9%, p<0.001) producedsignificant anti-tumor activity, but didn't exhibit any tumorregression. BCY6136 at 3 mg/kg, qw (TV=0 mm³, TGI=108.5%, p<0.001)completely eradicated the tumors, 1 of 5 tumors respectively in BCY61363 mg/kg groups showed regrowth after the dosing suspension and thetumors were resistant to BICY6136 treatment when resuming the dosing.The remaining tumors in the BCY6136 groups (4/5 for each group) showedno regrowth after 80 days of dosing suspension. In part 2 study, themean tumor size of vehicle treated mice reached 2342 mm³ on day 21 afterthe start of treatment. BCY6173 at 1 mg/kg, qw (TV=2151 mm³, TGI=8.9%,p>0.05) did not show anti-tumor antitumor activity. BCY6173 at 3 mg/kg,qw (TV=890 mm³, TGI=67.5%, p<0.05) produced obvious anti-tumor activity.

BCY6175 at 3 mg/kg, qw (TV=0 mm³, TGI=108.9%, p<0.001) completelyeradicated 4/5 tumors on day 14.

Study 15: In Vivo Efficacy Study of BCY6136 in the LU-01-0412 NSCLC PDXModel in Balb/c Nude Mice

(a) Study Objective

The objective of the project is to evaluate the in vivo therapeuticefficacy of BCY6136 in the LU-01-0412 NSCLC PDX model in BALB/c nudemice.

(b) Experimental Design

Dosing Dose Volume Dosing Gr Treatment n (mg/kg) (μl/g) Route Schedule 1Vehicle 6 — 10 iv Qw, 4 2 BCY6136 6 1 10 iv Qw, 4 3 BCY6136 6 3 10 ivQw, 4 4 BCY8245 6 3 10 iv Qw, 4 5 BCY8781 6 3 10 iv Qw, 4

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withLU-01-0412 tumor fragment (˜30 mm³) for tumor development. Animals wererandomized when the average tumor volume reached 159 mm³. The testarticle administration and the animal numbers in each group were shownin the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 25 mM Histidine 10%sucrose pH 7 BCY6136 1 Dissolve 6.06 mg BCY6136 in 5.969 ml 50 mMAcetate/acetic acid pH 5 10% sucrose 0.1 Dilute 180 μl 1 mg/ml BT5528with 1620 μl 50 mM Acetate/acetic acid pH 5 10% sucrose 0.3 Dilute 540μl 1 mg/ml BT5528 with 1260 ul 50 mM Acetate/acetic acid pH 5 10%sucrose BCY8245 1 Dissolve 4.15 mg BCY8245 powder in 4.121 ml vehiclebuffer 0.3 Dilute 540 μl 1 mg/ml BCY8245 with 1260 μl vehicle bufferBCY8781 1 Dissolve 4.08 mg BCY8781 powder in 80.8 μl DMSO, then diluteto 1 mg/ml with 3.958 vehicle buffer 0.3 Dilute 540 μl 1 mg/ml BCY8781with 1260 μl vehicle buffer

(iii) Sample Collection

Plasma from vehicle and 3 extra mice treated with BCY6136, BCY8245 andBCY8781 were collected at 30 min and 24 h post dosing. Tumor fromvehicle and 3 extra mice treated with BCY6136, BCY8245 and BCY8781 werecollected at 24 h post dosing.

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curves are shown in FIG. 16 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female BALB/c nude mice bearingLU-01-0412 xenograft is shown in Table 38.

TABLE 38 Tumor volume trace over time Group 1 Group 2 Group 3 Group 4Group 5 Vehicle BCY6136 BCY6136 BCY8245 BCY8781 Days Qw * 4 1 mpk, Qw *4 3 mpk, Qw * 4 3 mpk, Qw * 4 3 mpk, Qw * 4 0 159 ± 11 159 ± 13 159 ± 11159 ± 12 159 ± 11 4 255 ± 12 214 ± 16 197 ± 16 168 ± 18 176 ± 21 7 309 ±20 237 ± 16 195 ± 16 132 ± 10 167 ± 13 11 395 ± 31 246 ± 19 156 ± 18 78± 4 107 ± 15 14 464 ± 31 300 ± 18 177 ± 29 45 ± 5  72 ± 12 18 521 ± 26369 ± 32 210 ± 32 21 ± 2 44 ± 8 21 611 ± 33 470 ± 46 225 ± 32 11 ± 1 31± 6 25 737 ± 68 632 ± 47 252 ± 37  6 ± 1 20 ± 6 28 788 ± 80 664 ± 52 299± 37  2 ± 1 14 ± 5 32 1104 ± 142 758 ± 70 416 ± 52  1 ± 1 12 ± 5

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136, BCY8245 and BCY8781 in theLU-01-0412 xenograft model was calculated based on tumor volumemeasurements on day 32 after the start of the treatment.

TABLE 39 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GroupTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw * 4 1104 ± 142 — — — 2BCY6136, 1 mpk, 758 ± 70 68.6 36.7 p < 0.05  qw * 4 3 BCY6136, 3 mpk,416 ± 52 37.6 72.9 p < 0.001 qw * 4 4 BCY8245, 3 mpk,  1 ± 1 0.1 116.8 p< 0.001 qw * 4 5 BCY8781, 3 mpk, 12 ± 5 1.0 115.6 p < 0.001 qw * 4^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136, BCY8245 and BCY8781in the LU-01-0412 xenograft model was evaluated. The measured bodyweight and tumor volume of all treatment groups at various time pointsare shown in FIG. 16 and Tables 38 and 39.

The mean tumor volume of vehicle treated mice reached 1104 mm³ on day 32after the start of treatment. BCY6136 at 1 mg/kg, qw *4 (TV=758 mm³,TGI=36.7%, p<0.05) and 3 mg/kg, qw*4 (TV=416 mm³, TGI=72.9%, p<0.001)produced dose-dependent antitumor activity, but didn't show any tumorregression. BCY8245 at 3 mg/kg, qw*4 (TV=1 mm³, TGI=116.8%, p<0.001) andBCY8781 at 3 mg/kg, qw*4 (TV=12 mm³, TGI=115.6%, p<0.001) regressed thetumors obviously. Among them, 5 of 6 tumor treated with BCY8245 3 mg/kgand 2 of 6 tumor treated with d BCY8781 3 mg/kg were completelyeradicated on day 32.

In this study, animals in all groups maintained the body weight well.

Study 16: In Vivo Efficacy Study of BCY6136 in Treatment of LU-01-0486PDX Model in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in the LU-01-0486 PDX model in Balb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Gr Treatment n (mg/kg) (μl/g) Route Schedule 1Vehicle 5 — 10 iv qw 2 BCY6136 5 1 10 iv qw 3 BCY6136 5 2 10 iv qw 4BCY6136 5 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withLU-01-0486 of tumor fragment (˜30 mm³) for tumor development. Thetreatment was started when the average tumor volume reached 180 mm³ forefficacy study. The test article administration and the animal number ineach group are shown in the experimental design table.

(ii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10%sucrose pH 5 BCY6136 0.3 0.3 mg/ml BCY6136 was prepared as described inStudy 10 0.2 Dilute 940 μl 0.3 mg/ml BCY6136 with 470 μl Acetate buffer¹0.1 Dilute 470 μl 0.3 mg/ml BCY6136 with 940 μl Acetate buffer 1Acetatebuffer: 50 mM Acetate 10% sucrose pH 5

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 17 .

(ii) Tumor Volume Trace

Mean tumor volume on day 14 after the start of treatment in femaleBalb/c nude mice bearing LU-01-0486 xenograft is shown in Table 40.

TABLE 40 Tumor volume trace over time Days after the start of treatmentGroup Treatment 0 3 7 10 14 1 Vehicle, qw 179 ± 20 232 ± 30 358 ± 45 450± 47 651 ± 112 2 BCY6136, 180 ± 23 221 ± 20 326 ± 34 420 ± 34 638 ± 71 1 mpk, qw 3 BCY6136, 179 ± 27 222 ± 26 365 ± 44 459 ± 82 645 ± 105 2mpk, qw 4 BCY6136, 180 ± 25 209 ± 37 304 ± 51 348 ± 77 449 ± 115 3 mpk,qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the LU-01-0486 PDX model wascalculated based on tumor volume measurement at day 14 after the startof the treatment.

TABLE 41 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GroupTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 651 ± 112 — — — 2BCY6136, 1 mpk, qw 638 ± 71 98.0 3.0 p > 0.05 3 BCY6136, 2 mpk, qw 645 ±105 99.1 1.2 p > 0.05 4 BCY6136, 3 mpk, qw 449 ± 115 68.9 43.1 p > 0.05^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in LU-01-0486 PDXmodel was evaluated. The measured body weight and tumor volume of alltreatment groups at various time points are shown in the FIG. 17 andTables 40 and 41.

In this study, the mean tumor volume of vehicle treated mice reached 651mm³ on day 14 after the start of treatment. BCY6136 at 1 mg/kg, qw(TV=638 mm³, TGI=3.0%, p>0.05) and 2 mg/kg, qw (TV=645 mm³, TGI=1.2%,p>0.05) didn't show any anti-tumor activity. BCY6136 at 3 mg/kg, qw(TV=449 mm³, TGI=43.1%, p>0.05) produced slight anti-tumor activitywithout statistical significance.

Study 17: In Vivo Efficacy Test of BCY6136 in Treatment ofMDA-MB-231-Iuc Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of MDA-MB-231-luc xenograft model inBalb/c nude mice.

(b) Experimental Design

Dosing Dose Volume Dosing Gr Treatment n (mg/kg) (μl/g) Route Schedule 1Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10 iv qw 4BCY6136 3 2 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The cells growing in an exponential growth phase were harvested andcounted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withMDA-MB-231-luc tumor cells (10×10 {circumflex over ( )}6) in 0.1ml ofPBS with 0.1 ml matrigel for tumor development. 36 animals wererandomized when the average tumor volume reached 159 mm³. The testarticle administration and the animal numbers in each group were shownin the experimental design table.

(iii) Testing Article Formulation Preparation

Dose Treatment (mg/ml) Formulation Vehicle 50 mM Acetate, 10% sucrose pH= 5 BCY6136 1 Dissolve 3.79 mg BCY6136 into 3.695 ml formulation buffer0.3 Dilute 270 μl 1 mg/ml BCY6136 into 630 μl formulation buffer 0.2Dilute 180 μl 1 mg/ml BCY6136 into 720 μl formulation buffer 0.1 Dilute90 μl 1 mg/ml BCY6136 into 810 μl formulation buffer

(iv) Sample Collection

On PG-D33, we collected and fixed the tumors of Group 2 for FFPE.

At the end of study, we collected and fixed the tumors of Group 3 and 4for FFPE.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth are shown in FIG. 18 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearingMDA-MB-231-luc xenograft is shown in Tables 42 to 44.

TABLE 42 Tumor volume trace (PG-D 0~PG-D 17) Days after the start oftreatment Gr. Treatment 0 2 4 7 9 11 14 17 1 Vehicle, qw 159 ± 14 269 ±8  306 ± 19 425 ± 52 688 ± 54 908 ± 54 1064 ± 98  1315 ± 95  2 BCY6136,159 ± 10 226 ± 36 221 ± 54 310 ± 72 416 ± 89 526 ± 77 636 ± 92 809 ± 1351 mpk, qw 3 BCY6136, 159 ± 16 218 ± 17 182 ± 22 182 ± 26 101 ± 20  77 ±24 36 ± 4 41 ± 10 2 mpk, qw 4 BCY6136, 158 ± 5  241 ± 12 259 ± 6  325 ±14 258 ± 12 246 ± 15 162 ± 19 178 ± 10  3 mpk, qw

TABLE 43 Tumor volume trace (PG-D 19~PG-D 33) Days after the start oftreatment Gr. Treatment 19 21 24 26 28 31 33 1 Vehicle, qw 1453 ± 1281661 ± 173 — — — — — 2 BCY6136,  879 ± 190  994 ± 213 1253 ± 313 1431 ±353  1507 ± 253 2181 ± 609  — 1 mpk, qw 3 BCY6136, 35 ± 9 33 ± 9  31 ±17 41 ± 32  59 ± 45 82 ± 59 87 ± 71 2 mpk, qw 4 BCY6136, 171 ± 21 132 ±19 108 ± 19 85 ± 15 81 ± 8 87 ± 14 92 ± 18 3 mpk, qw

TABLE 44 Tumor volume trace (PG-D 35~PG-D 47) Days after the start oftreatment Gr. Treatment 35 38 40 42 45 47 3 BCY6136, 124 ± 106 156 ± 120179 ± 142 239 ± 197 285 ± 239 350 ± 298 2 mpk, qw 4 BCY6136, 129 ± 38 173 ± 65  181 ± 65  269 ± 113 293 ± 114 371 ± 128 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the MDA-MB-231-luc xenograftmodel was calculated based on tumor volume measurements at day 21 afterthe start of treatment.

TABLE 45 Tumor growth inhibition analysis Tumor Gr Treatment Volume(mm³)^(a) T/C^(b) (%) TGI (%) P value 1 Vehicle, qw 1661 ± 173 — — — 2BCY6136,  994 ± 213 59.8 44.4 p < 0.01  1 mpk, qw 3 BCY6136, 33 ± 9 2.0108.4 p < 0.001 2 mpk, qw 4 BCY6136, 132 ± 19 8.0 101.7 p < 0.001 3 mpk,qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the MDA-MB-231-lucxenograft model was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points are shown in theFIG. 18 and Tables 42 to 45.

The mean tumor size of vehicle treated mice reached 1661 mm³ on day 21.BCY6136 at 1 mg/kg (TV=994 mm³, TGI=44.4%, p<0.01) showed moderateantitumor activity, BCY6136 at 2 mg/kg (TV=33 mm³, TGI=108.4%, p<0.001)and 3 mg/kg (TV=132 mm³, TGI=101.1%, p<0.001) produced potent antitumoractivity, but the tumors showed obvious re-growth from day 28. In thisstudy, one mouse treated with BCY6136 2 mg/kg lost over 15% bodyweightduring the treatment schedule, other mice maintained the bodyweightwell.

Study 18: In Vivo Efficacy Test of BCY6136 in Treatment of EMT-6Syngeneic Model in BALB/c Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of EMT-6 syngeneic model in BALB/cmice.

(b) Experimental Design

Dose Dosing Sample Group Treatment (mg/kg) N Route Schedule Collection 1Vehicle — 5 iv qw * 4 tumors from 2 BCY6136 3 5 iv qw * 4 spare mice 3BCY6136  1/5^(b) 5 iv qw * 4 will be 4 BCY6136 0.3/3^(b) 5 iv qw * 4collected for FACS a. The injection volume of each mouse is 10 ml/kg.^(b)The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpkfrom Day 14.

(c) ExperimentaI Methods and Procedures

(i) Cell Culture

The EMT-6 tumor cells were maintained in vitro as a monolayer culture inEMEM medium supplemented with 10% heat inactivated fetal bovine serum at37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinelysubcultured twice weekly by trypsin-EDTA treatment.

The cells growing in an exponential growth phase were harvested andcounted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with EMT-6tumor cells (5×10⁶) in 0.1 ml of PBS for tumor development. 44 animalswere randomized when the average tumor volume reached 75 mm³. The testarticle administration and the animal numbers in each group were shownin the experimental design table.

(iii) Testing Article Formulation Preparation

BCY6136 formulation Conc. Treatment (mg/ml) Formulation Vehicle/buffer —50 mM Acetate, 10% sucrose pH = 5 BCY6136 1 Dissolve 6.2 mg BCY6136 with6113 ul buffer BCY6136 0.3 Dilute 450 μl 1 mg/ml BCY6136 stock with 1050μl buffer BCY6136 0.1 Dilute 150 μl 1 mg/ml BCY6136 stock with 1350 μlbuffer BCY6136 0.03 Dilute 45 μl 1 mg/ml BCY6136 stock with 1455 μlbuffer Vehicle/buffer — 50 mM Acetate, 10% sucrose pH = 5 BCY6136 1stock BCY6136 0.3 Dilute 420 μl 1 mg/ml BCY6136 stock with 980 μl bufferBCY6136 0.3 Dilute 420 μl 1 mg/ml BCY6136 stock with 980 μl bufferBCY6136 0.5 Dilute 700 μl 1 mg/ml BCY6136 stock with 700 μl buffer

(iv) Sample Collection

3 tumors from spare mice were collected for FACS on day 11. The data wassupplied by biology team.

(d) Results

(i) Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 19 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female BALB/c mice bearing EMT-6syngeneic is shown in Table 46.

TABLE 46 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 3 5 7 10 12 14 17 19 21 1 Vehicle, qw 82 ± 4 141 ± 11260 ± 24 443 ± 90 557 ± 99  703 ± 119 812 ± 139 948 ± 191 1129 ± 2481499 ± 340 2 BCY6136, 82 ± 4 58 ± 1 59 ± 2 125 ± 18 240 ± 23 322 ± 23374 ± 22  431 ± 37  486 ± 50 561 ± 61 3 mpk, qw 3 BCY6136, 82 ± 4 108 ±18 204 ± 27 350 ± 57 426 ± 49 588 ± 72 691 ± 65  850 ± 98  1018 ± 1151272 ± 140 1/5^(a) mpk, qw 4 BCY6136, 82 ± 4 130 ± 16 255 ± 35 358 ± 34450 ± 67 607 ± 94 731 ± 112 872 ± 119 1082 + 133 1394 ± 161 0.3/3^(a)mpk, qw The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpkfrom Day 14.

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in EMT-6 syngeneic model wascalculated based on tumor volume measurements on day 21 after the startof treatment.

TABLE 47 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compare Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw1499 ± 340 — — — 2 BCY6136, 3 mpk, qw 561 ± 61 37.4 66.2 p < 0.05 3BCY6136, 1/5^(c) mpk, 1272 ± 140 84.8 16.1 ns qw 4 BCY6136, 0.3/3^(c)1394 ± 161 93.0  7.4 ns mpk, qw ^(a)Mean ± SEM. ^(b)Tumor GrowthInhibition is calculated by dividing the group average tumor volume forthe treated group by the group average tumor volume for the controlgroup (T/C). ^(c)The dosage of group 3 and group 4 was changed to 5 mpkand 3 mpk from Day 14.

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in EMT-6 syngeneicmodel was evaluated. The measured body weights and tumor volumes of alltreatment groups at various time points are shown in the FIG. 19 andTables 46 and 47.

The mean tumor size of vehicle treated mice reached 1499 mm³ on day 21.BCY6136 at 3 mg/kg, qw (TV=561 mm³, TGI=66.2%, p<0.05) showed obviousantitumor activity. BCY6136 at 1/5 mg/kg, qw (TV=1272 mm³, TGI=16.1%,p>0.05) and BCY6136 at 0.3/3 mg/kg, qw (TV=1394 mm³, TGI=7.4%, p>0.05)didn't show any antitumor activity.

The dosage of group 3 and group 4 was changed to 5 mpk and 3 mpk fromday 14. Tumor ulceration was found in mouse 3-5 on Day 14, and the micewas deal with antibiotic cream. In this study, all mice maintained thebodyweight well.

Study 19: In Vivo Efficacy Study of BCY6136 in Treatment of NCI-N87Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of NCI-N87 xenograft in Balb/c nudemice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv Qw 2 BCY6136 3 1 10 iv Qw 3 BCY6136 3 2 10iv Qw 4 BCY6136 3 3 10 iv Qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The NCI-N87 tumor cells were maintained in RPMI-1640 medium supplementedwith 10% heat inactivated fetal bovine serum at 37° C. in an atmosphereof 5% CO₂ in air. The tumor cells were routinely subcultured twiceweekly. The cells growing in an exponential growth phase were harvestedand counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with NCI-N87tumor cells (10×10⁶) with matrigel (1:1) in 0.2 ml of PBS for tumordevelopment. The animals were randomized and treatment was started whenthe average tumor volume reached approximately 176 mm³. The test articleadministration and the animal number in each group are shown in theexperimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10%sucrose pH 5 BCY6136 1 Dissolve 4.295 mg BCY6136 in 4.214 ml Acetatebuffer¹ 0.1 Dilute 90 μl 1 mg/ml BCY6136 stock with 810 μl Acetatebuffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with 720 μl Acetatebuffer 0.3 Dilute 270 μl 1 mg/ml BCY6136 stock with 630 μl Acetatebuffer ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 20 .

(ii) Tumor Volume Trace

Mean tumor volume overtime in female Balb/c nude mice bearing NCI-N87xenograft is shown in Table 48.

TABLE 48 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 16 1 Vehicle, qw 174 ± 7 213 ± 5 266 ± 6 421 ± 10 537 ± 17 598 ± 30 734 ± 46 821 ± 55 2 BCY6136, 176 ± 7 200 ± 8210 ± 14 224 ± 27 238 ± 21 184 ± 18 244 ± 23 276 ± 35 1 mpk, qw 3BCY6136,  176 ± 18  197 ± 25 168 ± 25 170 ± 26 165 ± 34  96 ± 27 133 ±35 150 ± 52 2 mpk, qw 4 BCY6136, 177 ± 8 197 ± 9 169 ± 7  158 ± 3  148 ±8   95 ± 16 141 ± 12 145 ± 24 3 mpk, qw Days after the start oftreatment Gr. Treatment 18 21 23 25 28 30 1 Vehicle, qw 918 ± 91 1024 ±83  1151 ± 68  1305 ± 57  1407 ± 64  1465 ± 90  2 BCY6136, 308 ± 44 343± 37 390 ± 43 406 ± 48 422 ± 42 425 ± 47 1 mpk, qw 3 BCY6136, 160 ± 49190 ± 63 203 ± 65 218 ± 66 201 ± 53 210 ± 60 2 mpk, qw 4 BCY6136, 164 ±28 202 ± 28 205 ± 30 201 ± 16 196 ± 21 201 ± 22 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the NCI-N87 xenograft wascalculated based on tumor volume measurements at day 30 after the startof treatment.

TABLE 49 Tumor growth inhibition analysis Tumor Volume Group Treatment(mm³)^(a) T/C^(b) (%) TGI (%) P value 1 Vehicle, qw 1465 ± 90  — — — 2BCY6136, 425 ± 47 29.0 80.7 p < 0.001 1 mpk, qw 3 BCY6136, 210 ± 60 14.397.4 p < 0.001 2 mpk, qw 4 BCY6136, 201 ± 22 13.7 98.1 p < 0.001 3 mpk,qw ^(a)Mean ± SEM. ^(b)Tumor growth inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the NCI-N87 modelwas evaluated. The measured body weight and tumor volume of alltreatment groups at various time points are shown in the FIG. 20 andTables 48 and 49.

The mean tumor size of vehicle treated mice reached 1465 mm³ on day 30.BCY6136 at 1 mg/kg, qw (TV=425 mm³, TGI=80.7%, p<0.001) and 2 mg/kg, qw(TV=210 mm³, TGI=97.4%, p<0.001) produced significant antitumor activityin a dose-dependent manner, BCY6136 at 3 mg/kg, qw (TV=201 mm³,TGI=98.1%, p<0.001) showed comparable antitumor activity with BCY6136 at2 mpk.

In this study, no obvious body weight loss was found in all the groupsduring the treatment schedule.

Study 20: In Vivo Efficacy Study of BCY6136 in Treatment of SK-OV-3Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of SK-OV-3 xenograft in Balb/c nudemice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (ul/g) RouteSchedule 1 Vehicle 3 — 10 iv Qw 2 ADC 3 3 10 iv Qw 3 BCY6136 3 1 10 ivQw 4 BCY6136 3 2 10 iv Qw 5 BCY6136 3 3 10 iv Qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The SK-OV-3 tumor cells were maintained in McCoy's 5a mediumsupplemented with 10% heat inactivated fetal bovine serum at 37° C. inan atmosphere of 5% CO₂ in air. The tumor cells were routinelysubcultured twice weekly. The cells growing in an exponential growthphase were harvested and counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with SK-OV-3tumor cells (10×10⁶) with matrigel (1:1) in 0.2 ml of PBS for tumordevelopment. The animals were randomized and treatment was started whenthe average tumor volume reached approximately 186 mm³. The test articleadministration and the animal number in each group are shown in theexperimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article Purity (mg/ml) Formulation Vehicle — — 50 mM Acetate10% sucrose pH 5 BCY6136 98.5% 1 Dissolve 3.65 mg BCY6136 in 3.60 ml 50mM Acetate buffer¹ 0.1 Dilute 90 μl 1 mg/ml BCY6136 stock with 810 μlAcetate buffer¹ 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with 720 μlAcetate buffer¹ 0.3 Dilute 270 μl 1 mg/ml BCY6136 stock with 630 μlAcetate buffer¹ ADC ADC 0.3 Dilute 69 μl 10.47 mg/ml ADC stock with 2331μl ADC buffer² ¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5 ²ADCbuffer: 25 mM Histidine 10% sucrose pH 5.5

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 21 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing SK-OV-3xenograft is shown in Table 50.

TABLE 50 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 5 7 9 12 14 1 Vehicle, qw 187 ± 16 243 ± 24 313 ± 28399 ± 37 470 ± 23 606 ± 61  742 ± 103 2 ADC, 187 ± 16 181 ± 15 212 ± 16263 ± 35 268 ± 14 335 ± 23 353 ± 18 3 mpk, qw 3 BCY6136, 186 ± 23 222 ±19 293 ± 34 331 ± 21 356 ± 23 440 ± 8  503 ± 28 2 mpk, qw 4 BCY6136, 186± 23 170 ± 18 164 ± 28 188 ± 33 180 ± 34 202 ± 29 200 ± 29 2 mpk, qw 5BCY6136, 184 ± 24 168 ± 18 150 ± 12 164 ± 12 158 ± 8  180 ± 8  187 ± 4 3 mpk, qw Days after the start of treatment Gr. Treatment 16 19 21 23 2628 1 Vehicle, qw  891 ± 133 1076 ± 185 1173 ± 214 1340 ± 236 1490 ± 2731560 ± 305 2 ADC, 392 ± 63 449 ± 4  481 ± 27 573 ± 33 647 ± 26  684 ±111 3 mpk, qw 3 BCY6136, 587 ± 33 702 ± 43 752 ± 26 893 ± 34 1002 ± 68 1035 ± 67  2 mpk, qw 4 BCY6136, 230 ± 46 229 ± 48 231 ± 58 236 ± 49 240± 48 277 ± 58 2 mpk, qw 5 BCY6136, 212 ± 17 208 ± 29 204 ± 12 205 ± 17227 ± 31 254 ± 48 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the SK-OV-3 xenograft wascalculated based on tumor volume measurements at day 28 after the startof treatment.

TABLE 51 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GroupTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 1560 ± 305 — — — 2ADC, 3 mpk, qw  684 ± 111 43.9 63.8 p < 0.01  3 BCY6136, 1 mpk, qw 1035± 67  66.4 38.1 p > 0.05  4 BCY6136, 2 mpk, qw 277 ± 58 17.8 93.3 p <0.001 5 BCY6136, 3 mpk, qw 254 ± 48 16.3 95.0 p < 0.001 ^(a)Mean ± SEM.^(b)Tumor growth inhibition is calculated by dividing the group averagetumor volume for the treated group by the group average tumor volume forthe control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the SK-OV-3 modelwas evaluated. The measured body weight and tumor volume of alltreatment groups at various time points are shown in the FIG. 21 andTables 50 and 51.

The mean tumor size of vehicle treated mice reached 1560 mm³ on day 28.ADC at 3 mg/kg, qw (TV=684 mm³, TGI=63.8%, p<0.01) showed moderateanti-tumor efficacy. BCY6136 at 1 mg/kg, qw (TV=1035 mm³, TGI=38.1%,p>0.05) didn't show obvious anti-tumor activity. BCY6136 at 2 mg/kg, qw(TV=277 mm³, TGI=93.3%, p<0.001) and 3 mg/kg, qw (TV=254 mm³, TGI=95.0%,p<0.001) produced significant anti-tumor activity.

In this study, no obvious body weight loss was found in all the groupsduring the treatment schedule.

Study 21: In Vivo Efficacy Study of BCY6136 in Treatment of OE21Xenograft in Balb/c Nude Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of OE21 xenograft in Balb/c nude mice.

(b) Experimental Design

Dose Dosing Dosing Group Treatment n (mg/kg) Volume (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10iv qw 4 BCY6136 3 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The OE21 tumor cells were maintained in RPMI-1640 medium supplementedwith 10% heat inactivated fetal bovine serum at 37° C. in an atmosphereof 5% CO₂ in air. The tumor cells were routinely subcultured twiceweekly. The cells growing in an exponential growth phase were harvestedand counted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with OE21tumor cells (5×10⁶) with matrigel (1:1) in 0.2 ml of PBS for tumordevelopment. The animals were randomized and treatment was started whenthe average tumor volume reached approximately 157 mm³. The test articleadministration and the animal number in each group are shown in theexperimental design table.

(iii) Testing Article Formulation Preparation

Test Conc. article (mg/ml) Formulation Vehicle — 50 mM Acetate 10%sucrose pH 5 BCY6136 1 Dissolve 4.295 mg BCY6136 in 4.214 ml Acetatebuffer¹ 0.1 Dilute 90 μl 1 mg/ml BCY6136 stock with 810 μl Acetatebuffer 0.2 Dilute 180 μl 1 mg/ml BCY6136 stock with 720 μl Acetatebuffer 0.3 Dilute 270 μl mg/ml BCY6136 stock with 630 μl Acetate buffer¹Acetate buffer: 50 mM Acetate 10% sucrose pH 5

(d) Results

Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve is shown in FIG. 22 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing OE21xenograft is shown in Table 52.

TABLE 52 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 1 Vehicle, qw 155 ± 9  211 ± 16 291 ± 16 379± 14 456 ± 32 539 ± 13 2 BCY6136, 159 ± 14 202 ± 28 251 ± 29 282 ± 6 331 ± 19 392 ± 35 1 mpk, qw 3 BCY6136, 157 ± 19 197 ± 13 219 ± 6  235 ±27 268 ± 35 243 ± 37 2 mpk, qw 4 BCY6136, 155 ± 19 200 ± 16 197 ± 7  209± 11 229 ± 26 211 ± 14 3 mpk, qw Days after the start of treatment Gr.Treatment 14 16 18 21 23 1 Vehicle, qw 828 ± 42 955 ± 40 1035 ± 58  1250± 46 1586 ± 57 2 BCY6136, 609 ± 56 694 ± 44 777 ± 68 1083 ± 85 1155 ± 981 mpk, qw 3 BCY6136, 346 ± 78 371 ± 98  396 ± 109  515 ± 94  537 ± 122 2mpk, qw 4 BCY6136, 289 ± 38 318 ± 53 330 ± 40  474 ± 42  489 ± 51 3 mpk,qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the OE21 xenograft wascalculated based on tumor volume measurements at day 23 after the startof treatment.

TABLE 53 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GroupTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 1586 ± 57 — — — 2BCY6136, 1 mpk, qw 1155 ± 98 72.8 30.4 p < 0.05  3 BCY6136, 2 mpk, qw 537 ± 122 33.9 73.4 p < 0.001 4 BCY6136, 3 mpk, qw  489 ± 51 30.8 76.7p < 0.001 ^(a)Mean ± SEM. bTumor growth inhibition is calculated bydividing the group average tumor volume for the treated group by thegroup average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the OE21 model wasevaluated. The measured body weight and tumor volume of all treatmentgroups at various time points are shown in the FIG. 22 and Tables 52 and53.

The mean tumor size of vehicle treated mice reached 1586 mm³ on day 23.BCY6136 at 1 mg/kg, qw (TV=1155 mm³, TGI=30.4% p<0.05) showed slightanti-tumor activity. BCY6136 at 2 mg/kg, qw (TV=537 mm³, TGI=73.4%,p<0.001) and 3 mg/kg, qw (TV=489 mm³, TGI=76.7%, p<0.001) producedsignificant anti-tumor activity.

In this study, no obvious body weight loss was found in all the groupsduring the treatment schedule.

Study 22: In Vivo Efficacy Test of BCY6136 in Treatment of MOLP-8Xenograft in CB17-SCID Mice

(a) Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY6136 in treatment of MOLP-8 xenograft in CB17-SCID mice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv qw 2 BCY6136 3 1 10 iv qw 3 BCY6136 3 2 10iv qw 4 BCY6136 3 3 10 iv qw

(c) Experimental Methods and Procedures

(i) Cell Culture

The MOLP-8 tumor cells were maintained in vitro as a monolayer culturein RMPI-1640 supplemented with 20% heat inactivated fetal bovine serumat 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells wereroutinely subcultured by trypsin-EDTA treatment. The cells growing in anexponential growth phase were harvested and counted for tumorinoculation.

(ii) Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with MOLP-8tumor cells (10×10⁶) in 0.2 ml PBS with 50% matrigel for tumordevelopment. 36 animals were randomized when the average tumor volumereached 141 mm³. The test article administration and the animal numbersin each group were shown in the experimental design table.

(iii) Testing Article Formulation Preparation

Concentration Treatment (mg/ml) Formulation Vehicle — 50 mM Acetate, 10%sucrose pH = 5 BCY6136 0.1 Dilute 90 μl 1 mg/ml BCY6136 stocks* with 810μl buffer** 0.2 Dilute 180 μl 1 mg/ml BCY6136 stocks* with 720 μlbuffer** 0.3 Dilute 270 μl 1 mg/ml BCY6136 stocks* with 630 μl buffer***BCY6136 stocks: 10.93 mg BCY6136 dissolved to 10.93 mL 50 mM Acetate,10% sucrose, pH = 5, and separated into individual tubes and stored at−80° C. **Buffer: 50 mM Acetate, 10% sucrose pH = 5

(d) Results

(i) Body Weight change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIG. 23 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female CB17-SCID mice bearing MOLP-8xenograft is shown in Table 54.

TABLE 54 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 139 ± 2  375 ± 36 604 ± 28984 ± 88 1451 ± 133 1981 ± 196 2528 ± 295 2 BCY6136, 143 ± 13 299 ± 6 444 ± 49 576 ± 31 806 ± 85 1132 ± 170 1446 ± 234 1 mpk, qw 3 BCY6136,140 ± 15 271 ± 43 250 ± 2  509 ± 23 662 ± 78 873 ± 49 1218 ± 144 2 mpk,qw 4 BCY6136, 142 ± 19 239 ± 67 197 ± 20 342 ± 78 425 ± 90  693 ± 133 938 ± 155 3 mpk, qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY6136 in the MOLP-8 xenograft modelwas calculated based on tumor volume measurements at day 14 after thestart of treatment.

TABLE 55 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw2528 ± 295 — — — 2 BCY6136, 1 1446 ± 234 57.2 45.5 p > 0.05 mpk, qw 3BCY6136, 2 1218 ± 144 48.2 54.9 p < 0.05 mpk, qw 4 BCY6136, 3  938 ± 15537.1 66.7 p < 0.01 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibitionis calculated by dividing the group average tumor volume for the treatedgroup by the group average tumor volume for the control group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCY6136 in the MOLP-8xenograft model was evaluated. The measured body weights and tumorvolumes of all treatment groups at various time points are shown in theFIG. 23 and Tables 54 and 55.

The mean tumor size of vehicle treated mice reached 2528 mm³ on day 14.BCY6136 at 1 mg/kg (TV=1146 mm³, TGI=45.5%, p>0.05), 2 mg/kg (TV=1218mm³, TGI=54.9%, p<0.05) and 3 mg/kg (TV=938 mm³, TGI=66.7%, p<0.01)produced dose-dependent antitumor activity, but all of dosage didn'tregress the tumors in MOLP-8 xenografts. In this study, all of micemaintained the bodyweight well.

Study 23: In Vivo Efficacy Test of BCYs in Treatment of HT1080 Xenograftin BALB/c Nude Mice

(a) Study Objective

The objective of the research was to evaluate the in vivo anti-tumorefficacy of BCYs in treatment of HT1080 xenograft model in BALB/c nudemice.

(b) Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv qw 2 BCY6173 3 1 10 iv qw 3 BCY6173 3 2 10iv qw 4 BCY6173 3 3 10 iv qw 5 BCY6135 3 1 10 iv qw 6 BCY6135 3 2 10 ivqw 7 BCY6135 3 3 10 iv qw 8 BCY6136 3 2 10 iv qw 9 BCY6136 3 3 10 iv qw10 BCY6136 3 5 10 iv qw 11 BCY6174 3 1 10 iv qw 12 BCY6174 3 2 10 iv qw13 BCY6174 3 3 10 iv qw 14 BCY6175 3 1 10 iv qw 15 BCY6175 3 2 10 iv qw16 BCY6175 3 3 10 iv qw 17 ADC 3 3 10 iv qw Note: n: animal number;Dosing volume: adjust dosing volume based on body weight 10 μl/g.

(c) Experimental Methods and Procedures

Cell Culture

The HT1080 tumor cells will be maintained in medium supplemented with10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5%CO2 in air. The tumor cells will be routinely subcultured twice weekly.The cells growing in an exponential growth phase will be harvested andcounted for tumor inoculation.

(ii) Tumor Inoculation

Each mouse will be inoculated subcutaneously at the right flank withHT1080 tumor cells (5*10⁶) for tumor development. The animals will berandomized and treatment will be started when the average tumor volumereaches approximately 150-200 mm³. The test article administration andthe animal numbers in each group are shown in the following experimentaldesign table.

(iii) Testing Article Formulation Preparation

Dose Treatment (mg/ml) Formulation Vehicle — 50 mM Acetate/acetic acidpH 5 10% sucrose BCY6173 1 Dissolve 2.13 mg BCY6173 with 2.04 ml buffer0.1 Dilute 90 μl 1 mg/ml BCY6173 stock with 810 μl buffer 0.2 Dilute 180μl 1 mg/ml BCY6173 stock with 720 μl buffer 0.3 Dilute 270 μl 1 mg/mlBCY6173 stock with 630 μl buffer BCY6135 1 Dissolve 2 mg BCY6135 with1.9 ml buffer 0.1 Dilute 90 μl 1 mg/ml BCY6135 stock with 810 μl buffer0.2 Dilute 180 μl 1 mg/ml BCY6135 stock with 720 μl buffer 0.3 Dilute270 μl 1 mg/ml BCY6135 stock with 630 μl buffer BCY6136 0.2 Dilute 200μl 1 mg/ml BCY6136 stock with 800 μl buffer 0.3 Dilute 300 μl 1 mg/mlBCY6136 stock with 700 μl buffer 0.5 Dilute 500 μl 1 mg/ml BCY6136 stockwith 500 μl buffer BCY6174 1 Dissolve 2.69 mg BCY6174 with 2.677 mlbuffer 0.1 Dilute 90 μl 1 mg/ml BCY6174 stock with 810 μl buffer 0.2Dilute 180 μl 1 mg/ml BCY6174 stock with 720 μl buffer 0.3 Dilute 270 μl1 mg/ml BCY6174 stock with 630 μl buffer BCY6175 1 Dissolve 2 mg BCY6175with 1.924 ml buffer 0.1 Dilute 90 μl 1 mg/ml BCY6175 stock with 810 μlbuffer 0.2 Dilute 180 μl 1 mg/ml BCY6175 stock with 720 μl buffer 0.3Dilute 270 μl 1 mg/ml BCY6175 stock with 630 μl buffer ADC 0.3 Dilute25.78 μl 10.47 mg/ml ADC stock with 874.22 μl 25 mM Histidine pH 7 10%sucrose

(d) Results

Body Weight Change and Tumor Growth Curve

Body weight and tumor growth curve are shown in FIGS. 24 to 29 .

(ii) Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing HT1080xenograft is shown in Table 56.

TABLE 56 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 179 ± 22 312 ± 84  529 ± 135 886 ± 207 1185 ± 172  1467 ± 224  1737 ± 258 2 BCY6173 178 ± 26 276 ±8  328 ± 73 594 ± 62 745 ± 22  960 ± 53  1074 ± 150 1 mpk, qw 3 BCY6173,178 ± 28 277 ± 61  262 ± 125  309 ± 238 425 ± 334 436 ± 323  480 ± 347 2mpk, qw 4 BCY6173, 179 ± 43 182 ± 71 133 ± 88  87 ± 68 77 ± 65 60 ± 54 47 ± 42 3 mpk, qw 5 BCY6135 178 ± 22 267 ± 66 262 ± 58 436 ± 67 599 ±89  703 ± 36  871 ± 28 1 mpk, qw 6 BCY6135 178 ± 23 176 ± 48 117 ± 43 70 ± 23 67 ± 23 52 ± 21 62 ± 7 2 mpk, qw 7 BCY6135 177 ± 39 178 ± 79 92 ± 67  62 ± 46 62 ± 51 57 ± 51  44 ± 40 3 mpk, qw 8 BCY6136 178 ± 19249 ± 22 115 ± 8  126 ± 53 158 ± 71  140 ± 89   245 ± 116 2 mpk, qw 9BCY6136 178 ± 36 168 ± 21  72 ± 18 22 ± 7 21 ± 15 8 ± 6  3 ± 2 3 mpk, qw10 BCY6136 178 ± 26 165 ± 33  52 ± 10 18 ± 7 9 ± 4 5 ± 2  2 ± 1 5 mpk,qw 11 BCY6174 180 ± 35 231 ± 19 226 ± 29 432 ± 37 602 ± 63  742 ± 62 1066 ± 130 1 mpk, qw 12 BCY6174 178 ± 31 203 ± 50 123 ± 29 216 ± 47 291± 40  326 ± 68  532 ± 91 2 mpk, qw 13 BCY6174 178 ± 33 195 ± 13 110 ± 39 58 ± 23 34 ± 17 21 ± 11 11 ± 7 3 mpk, qw 14 BCY6175 178 ± 27 248 ± 62244 ± 74 347 ± 18 435 ± 18  558 ± 38  769 ± 26 1 mpk, qw 15 BCY6175 178± 22 223 ± 42 158 ± 59 116 ± 35 156 ± 52  166 ± 51  295 ± 88 2 mpk, qw16 BCY6175 179 ± 39 189 ± 48 116 ± 50  43 ± 18 33 ± 18 25 ± 13 11 ± 9 3mpk, qw 17 ADC 180 ± 26 158 ± 30 58 ± 8 18 ± 2 7 ± 1 2 ± 2  0 ± 0 3 mpk,qw

(iii) Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCYs in the HT1080 xenograft model wascalculated based on tumor volume measurements at day 14 after the startof treatment.

TABLE 57 Tumor growth inhibition analysis Tumor Volume TGI P value GrTreatment (mm³)^(a) T/C^(b) (%) (%) compare 1 Vehicle, qw 1737 ± 258 — —— 2 BCY6173, 1 mpk, 1074 ± 150 61.8 42.5 p > 0.05  3 BCY6173, 2 mpk, 480 ± 347 27.6 80.6 p < 0.05  4 BCY6173, 3 mpk,  47 ± 42 2.7 108.4 p <0.01  5 BCY6135, 1 mpk, 871 ± 28 50.1 55.5 p < 0.01  6 BCY6135, 2 mpk,62 ± 7 3.5 107.5 p < 0.001 7 BCY6135, 3 mpk,  44 ± 40 2.5 108.6 p <0.001 8 BCY6136, 2 mpk, qw  245 ± 116 14.1 95.7 p < 0.001 9 BCY6136, 3mpk,  3 ± 2 0.2 111.2 p < 0.001 10 BCY6136, 5 mpk,  2 ± 1 0.1 111.3 p <0.001 11 BCY6174, 1 mpk, 1066 ± 130 61.4 43.1 p < 0.05  12 BCY6174, 2mpk, qw 532 ± 91 30.6 77.3 p < 0.01  13 BCY6174, 3 mpk, 11 ± 7 0.6 110.7p < 0.001 14 BCY6175, 1 mpk, 769 ± 26 44.3 62.1 p < 0.01  15 BCY6175, 2mpk, 295 ± 88 17.0 92.5 p < 0.001 16 BCY6175, 3 mpk, 11 ± 9 0.6 110.8 p< 0.001 17 ADC, 3 mpk, qw  0 ± 0 0.0 111.5 — ^(a)Mean ± SEM. ^(b)TumorGrowth Inhibition is calculated by dividing the group average tumorvolume for the treated group by the group average tumor volume for thecontrol group (T/C).

(e) Results Summary and Discussion

In this study, the therapeutic efficacy of BCYs in the HT1080 xenograftmodel was evaluated. The measured body weights and tumor volumes of alltreatment groups at various time points are shown in the FIGS. 24 to 29and Tables 56 and 57.

The mean tumor size of vehicle treated mice reached 1737 mm³ on day 14.

BCY6173 at 1 mg/kg, qw (TV=1074 mm³, TGI=42.5%, p>0.05), 2 mg/kg, qw(TV=480 mm³,

TGI=80.6%, p<0.05) and 3 mg/kg, qw (TV=7 mm³, TGI=108.4%, p<0.01)produced dose-dependent antitumor activity.

BCY6135 at 1 mg/kg, qw (TV=871 mm³, TGI=55.5%, p<0.01), 2 mg/kg, qw(TV=62 mm³, TGI=107.5%, p<0.001) and 3 mg/kg, qw (TV=44 mm³, TGI=108.6%,p<0.001) produced dose-dependent antitumor activity.

BCY6136 at 2 mg/kg, qw (TV=345 mm³, TGI=95.7%, p<0.001), 3 mg/kg, qw(TV=3 mm³, TGI=111.2%, p<0.001) and 5 mg/kg, qw (TV=2 mm³, TGI=111.3%,p<0.001) showed potent anti-tumor activity.

BCY6174 at 1 mg/kg, qw (TV=1066 mm³, TGI=43.1%, p<0.05), 2 mg/kg, qw(TV=532 mm³, TGI=77.3%, p<0.01) and 3 mg/kg, qw (TV=11 mm³, TGI=110.7°/0, p<0.001) produced dose-dependent antitumor activity.

BCY6175 at 1 mg/kg, qw (TV=769 mm³, TGI=62.1%, p<0.01), 2 mg/kg, qw(TV=295 mm³, TGI=92.5%, p<0.001) and 3 mg/kg, qw (TV=11 mm³, TGI=110.8°/0, p<0.001) produced dose-dependent antitumor activity.

ADC at 3 mg/kg, qw (TV=0 mm³, TGI=111.5%) completely eradicated thetumors by day 14.

1-21. (canceled)
 22. A method for preventing, suppressing, or treating adisease or disorder characterized by overexpression of EphA2 in diseasedtissue in a patient, the method comprising administering to the patienta peptide ligand specific for EphA2 comprising a polypeptide comprisingthree cysteine residues, separated by two loop sequences, and anon-aromatic molecular scaffold which forms covalent bonds with thecysteine residues of the polypeptide, such that two polypeptide loopsare formed on the molecular scaffold, wherein the peptide ligandcomprises the amino acid sequence: (SEQ ID NO: 1)C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii);

wherein HyP is hydroxyproline, HArg is homoarginine, and C₁, C_(ii), andC_(iii) represent first, second, and third cysteine residues,respectively, or a pharmaceutically acceptable salt thereof.
 23. Themethod of claim 22, wherein the molecular scaffold is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).
 24. Themethod of claim 22, wherein the peptide ligand comprises the amino acidsequence: (SEQ ID NO: 2)(β-Ala)-Sar₁₀-A(HArg)D-C_(i)(HyP)LVNPLC_(ii)LHP(D-Asp)W(HArg)C_(iii) (BCY6099);

wherein Sar is sarcosine, HArg is homoarginine and HyP ishydroxyproline.
 25. The method of claim 22, wherein the peptide ligandis in the form of a free acid.
 26. The method of claim 22, wherein thepharmaceutically acceptable salt is selected from a sodium salt, apotassium salt, a calcium salt, and an ammonium salt.
 27. The method ofclaim 22, wherein the EphA2 is human EphA2.
 28. The method of claim 22,wherein the peptide ligand is conjugated to one or more effector and/orfunctional groups to form a drug conjugate.
 29. The method of claim 28,wherein the effector and/or functional group is a cytotoxic agent. 30.The method of claim 29, wherein the cytotoxic agent is MMAE.
 31. Themethod of claim 29, wherein the drug conjugate comprises a linkerbetween the peptide ligand and the cytotoxic agent.
 32. The method ofclaim 31, wherein the cytotoxic agent is IVIMAE and the linker isselected from: -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-,-Ala-Ala-Asn-, D-Ala-Phe-Lys-, and -Glu-Pro-Cit-Gly-hPhe-Tyr-Leu- (SEQID NO: 3).
 33. The method of claim 32, wherein the cytotoxic agent isIVIMAE and the linker is -Val-Cit-.
 34. The method of claim 31, whereinthe cytotoxic agent is DM1 and the linker is selected from: -S-S-,-SS(SO₃H)-, -SS-(Me)-, -(Me)-SS-(Me)-, -SS-(Me₂)- and -SS-(Me)-SO₃H-.35. The method of claim 34, wherein the cytotoxic agent is DM1 and thelinker is selected from: -S-S- and -SS(SO₃H)-.
 36. The method of claim28, wherein the drug conjugate is selected from BCY6027, BCY6028,BCY6135, BCY6136, BCY6173, BCY6174, and BCY6175.
 37. The method of claim28, wherein the drug conjugate is selected from BCY6135, BCY6136,BCY6173, BCY6174, and BCY6175.
 38. The method of claim 28, wherein thedrug conjugate is BCY6136.
 39. The method of claim 22, wherein thedisease or disorder is cancer.
 40. The method of claim 39, wherein thecancer is selected from prostate cancer, lung cancer, breast cancer,gastric cancer, ovarian cancer, oesophageal cancer, multiple myeloma,and fibrosarcoma.
 41. The method of claim 22, wherein the patient isidentified as having an increased copy number variation (CNV) of EphA2.